Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys⁷³ only after the formation of a Cys³²-Cys³⁵ disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1^C32S/C35S (a mutant Trx1 with both Cys³² and Cys³⁵ replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys¹⁷³ and Cys⁸³ and protects it from H₂O₂-induced overoxidation. Functionally, we found that Cys⁷³-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.Nitric oxide (NO) is an important second messenger for signal transduction in cells. The production of cGMP by guanylyl cyclase, enabled by the binding of NO onto heme, is considered the primary mechanism responsible for the plethora of functions exerted by NO (1). However, S-nitrosylation, the covalent addition of the NO moiety onto cysteine thiols, is increasingly recognized as an important post-translational modification for regulating protein functions (for reviews, see Refs. 2 and 3). S-Nitrosylation is dynamic, reversible, site-specific, and modulated by selected cellular stimuli (4–7). With improved detection sensitivity, an increasing number of S-nitrosylated proteins have been identified by proteomics technologies (5, 8–13). Among the known modified proteins, nitrosylation occurs only on selected cysteines (4, 6, 14–17). Non-enzymatic mechanisms proposed to determine S-nitrosylation specificity include the availability of specific NO donors and protein microenvironments that stabilize the pK_a of acidic target cysteines (18). Furthermore, several enzymes, including hemoglobin (19, 20), superoxide dismutase 1 (21, 22), S-nitrosoglutathione reductase (23–25), and protein-disulfide isomerase (26), have been shown to possess either transnitrosylase or denitrosylase activities. However, an enzymatic system that governs site-specific transnitrosylation and denitrosylation, analogous to the kinase/phosphatase paradigm for regulating protein phosphorylation, has remained largely uncharacterized.Trx1¹ is an important antioxidant protein with protein reductase activity (27, 28). It has been characterized as an antiapoptotic protein because of its ability to suppress proapoptotic proteins, including apoptosis signal-regulating kinase 1 via disulfide reduction and Casp3 via transnitrosylation of Cys¹⁶³ (14, 29). Conversely, Trx1 can denitrosylate Casp3 at Cys¹⁶³, resulting in Casp3 activation (7). Trx1 appears to govern site-specific reversible nitrosylation of selected protein targets (14, 15), but what are the underlying mechanisms that regulate Trx1 transnitrosylation and denitrosylation activities? Are there additional Trx1-mediated transnitrosylation or denitrosylation targets that have not yet been identified? In this study, we used ESI-Q-TOF mass spectrometry (MS) to analyze the nitrosylation of Trx1 and a Casp3 peptide (Casp3p) under different redox conditions. Because of the labile nature of the S–NO bond, direct identification of S-nitrosylated proteins and their specific nitrosylation sites by MS remains challenging (8). A biotin switch method that is based on the derivatization of protein S–NO with a biotinylating agent is typically used for such analyses (8). However, like any indirect method, both false positive and negative identifications have been reported (30). Recently, we developed a method for direct analysis of protein S-nitrosylation by ESI-Q-TOF MS without prior chemical derivatization (31). Here we applied the same technique to determine the regulation of Trx1 by stepwise oxidative and nitrosative modifications of distinct cysteines and its subsequent ability to transnitrosylate target proteins. Nitrosative modification at Cys⁷³ of Trx1 cannot occur without prior attenuation of the Trx1 disulfide reductase and denitrosylase activities via either disulfide bond formation between Cys³² and Cys³⁵ or their mutation to serines. This is a key observation that has never been previously reported. Consequently, we designed a proteomics approach and discovered over 40 putative Trx1 transnitrosylation target proteins. We further characterized the Trx1 transnitrosylation proteome and identified three consensus motifs surrounding the putative Trx1 transnitrosylation sites, suggesting a protein-protein interaction mechanism for determining transnitrosylation specificity.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX₁₆C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX₁₆C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur.Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1–4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4–6). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the “small Tim” and Cox17 families (7–10). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11–13).The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1α and Ero1β) are large flavoenzymes present in the ER with at least five disulfide bonds, but only two of the disulfide bonds are conserved. The conserved cysteines are essential for the catalytic activity of Ero1p forming the active site CXXC and shuttle disulfide CX₄C, respectively (15, 16). Furthermore, nonconserved disulfide bonds have been shown recently to be important in regulating the activity of both yeast and mammalian Ero1 (17–19). The second group of oxidases, the multidomain quiesin sulfhydryl oxidases, have important functions in higher eukaryotes (14, 20). Quiesin sulfhydryl oxidases consist of an Erv/ALR module fused to one or more thioredoxin-like domains with two conserved CXXC motifs in the Erv/ALR module. Quiesin sulfhydryl oxidase enzymes are found in many subcellular and extracellular locations, but not in mitochondria. Instead, single domain Erv/ARL enzymes of the third group are found in the 7mitochondria of many eukaryotic cells (21). Erv1p belongs to this single domain Erv/ARL family, which includes the human mitochondrial ARL, plant AtErv1, and yeast Erv2p of the ER lumen.The Erv/ARL enzymes are characterized by a highly conserved central catalytic core of ∼100 amino acids, which includes an active site CXXC motif (Cys¹³⁰–Cys¹³³ for Erv1p), CX₁₆C disulfide bond (Cys¹⁵⁹–Cys¹⁷⁶ for Erv1p), and residues involved in FAD binding (Fig. 1A). Based on the partial crystal structure data of Erv2p (22) and AtErv1 (23), the catalytic core of Erv proteins contains a four-helix bundle forming the noncovalent FAD-binding site with the active site CXXC in close proximity to the isoalloxazine ring of FAD. In addition, the long range CX₁₆C disulfide bond of the Erv proteins brings the short fifth helix to the four-helix bundle in proximity to the adenine ring of FAD (Fig. 1A). Thus, the CX₁₆C disulfide bond is proposed to play a structural role in stabilizing the FAD binding and/or protein folding, but direct experimental evidence to verify the roles is lacking. Apart from the catalytic core, the other parts of the proteins seem flexible and unfolded. Importantly, all members of the Erv/ALR family have at least an additional disulfide bond located in the nonconserved N- or C-terminal region to the catalytic core (Fig. 1B), which is hypothesized as a shuttle disulfide based on the partial crystal structure of Erv2 (22). The hypothesized shuttle disulfide of Erv2p CXC and AtErv1 CX₄C are located in the C terminus, but Erv1p (Cys³⁰–Cys³³) and ALR have a CXXC shuttle disulfide located N-terminal to the catalytic core. Furthermore, structural and chemical data have suggested that Erv/ARL enzymes form homodimer or oligomers in the presence or absence of intermolecular disulfide bonds (5, 23, 24).Open in a separate windowFIGURE 1.Structure and conserved Cys motifs of Erv/ALR enzymes. A, modeled structures of the conserved central catalytic core domain of Erv1p dimer based on the crystal structure data of AtErv1 (Protein Data Bank accession number 2HJ3, residues 73–173, the helix 1 starts with residue 75). The helices of the four-helix bundle and the short fifth helix are labeled from 1 to 5. The two disulfides are shown as yellow spheres, and the cofactor FAD is in red. The Cys¹³⁰–Cys¹³³ is the redox active disulfide located closely to the isoalloxazine ring of FAD. The N and C termini were labeled as N and C, respectively. The structure was generated using Pymol program. B, schematic of the primary structure of yeast, plant, and human sulfhydryl oxidase with the conserved Cys motifs. The conserved central catalytic core regions are shown as black bars, and the nonconserved regions are in gray.Yeast mitochondrial Erv1p contains a total of six Cys residues forming three pairs of disulfide bonds (residues 30–33, 130–133, and 159–176) as described above. Previous studies with single Cys mutants showed that although all three disulfide bonds are essential for Erv1p function in vivo, only Cys¹³⁰–Cys¹³³ disulfide is required for the oxidase activity of Erv1p in vitro (24). The conclusion that only Cys¹³⁰–Cys¹³³ disulfide is required for Erv1p oxidase activity in vitro was based on a study using the artificial substrate DTT² as the electron donor. Abnormal color changes were observed for some of the single Cys mutants of Erv1p in the previous study that were probably caused by protein misfolding or formation of non-native disulfides because of the presence of a redox active but unpaired Cys. It is clear that Cys¹³⁰–Cys¹³³ is the active site disulfide; however, experimental evidence for the role of Cys³⁰–Cys³³ disulfide is lacking, and the specific role played by the unique CX₁₆C motif of Erv proteins is unknown.In this study, we dissected the structural and functional roles of all three individual disulfides of Erv1p systematically. To avoid misfolding via unpaired Cys, three double Cys mutants of Erv1p were generated with each of the disulfides mutated to serines. All three mutants were successfully purified with the normal FAD binding properties of the wild type (WT) Erv1p. Various biophysical and biochemical methods were used to study the folding and oxidase activity of the WT and Erv1p mutants. Both artificial and the natural substrate (Mia40) of Erv1p were used as electron donors to understand the functional mechanism of Erv1p. Our results show that both the first (Cys³⁰–Cys³³) and second (Cys¹³⁰–Cys¹³³) disulfides are essential for Erv1 oxidase activity in vitro. Although none of the three disulfides are essential for FAD binding, the third disulfide (Cys¹⁵⁹–Cys¹⁷⁶) plays an important role in stabilizing the folding of Erv1p. More importantly, this study provided direct experimental evidence to show that Cys³⁰–Cys³³ functionally acts as a shuttle disulfide passing electrons to the active site Cys¹³⁰–Cys¹³³ disulfide. Moreover, the electron transfer seems to occur through both intersubunit and intermolecular interactions.     

The Ternary Structure of the Double-headed Arrowhead Protease Inhibitor API-A Complexed with Two Trypsins Reveals a Novel Reactive Site Conformation

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 Å resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 Å apart. The overall fold of API-A belongs to the β-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu⁸⁷ and Lys¹⁴⁵, and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met⁸³ to P5′ Ala⁹², adopts a novel conformation with the Leu⁸⁷ completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys¹⁴¹ to P5′ Glu¹⁵⁰, binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.Protease inhibitors (PIs)⁴ are ubiquitously distributed in all organisms, including plants, animals, and microorganisms (1). They play vital roles in regulating their corresponding proteases, which are involved in many biological processes such as protein digestion, cell signal transmission, inflammation, apoptosis, blood coagulation, and embryogenesis (2). The clinical applications of PIs are widespread, and there is great interest in developing more potent therapeutic PIs for treating human diseases related to cancer (3), pancreatitis (4), thrombosis (5), and AIDS (6). To this end, the soybean Kunitz-type serine proteases inhibitors have been extensively studied (1, 7–11). The inhibitors of this family generally contain 170–200 residues and have two disulfide bonds. Most members have only one reactive site located in the region of residues 60–70 (7, 10, 12–14). However, a few members possess two reactive sites that simultaneously bind two protease molecules and are thus termed double-headed inhibitors (15–18). All of these inhibitors are classified into family I3 of peptidase inhibitors (19). Most members are further grouped into subfamily I3A. However, the double-headed arrowhead PIs API-A and -B are grouped in subfamily I3B because of their very low sequence similarity to other members (19). In contrast to other double-headed PIs such as the Bowman-Birk and ovomucoid inhibitors, which have two identical reactive sites that have evolved by domain shuffling and gene duplication (1, 20–25), both API-A and -B have two distinct reactive sites.API-A and -B were first purified from the tubers of Sagittaria sagittifolia (Linn) in 1979 (26). Both consist of 179 residues with three disulfide bonds and can inhibit a variety of serine proteases, including trypsin, chymotrypsin, and porcine tissue kallikrein (17, 26–28). Although the sequence identity of API-A and -B is as high as 91%, their inhibitory specificities differ. The former can bind one molecule of trypsin and one molecule of chymotrypsin, whereas the latter can simultaneously bind two molecules of trypsin (26). The two P1 residues of the reactive sites of API-A and -B were first predicted to be Lys⁴⁴ and Arg⁷⁶ based on their surrounding sequences, which are similar to those of the reactive sites of bovine pancreas trypsin inhibitor and soybean Kunitz trypsin inhibitor (29). However, their identities were later revised to Arg⁷⁶ and Leu⁸⁷ (for API-A) or Lys⁸⁷ (for API-B) based on results from sited-directed mutagenesis studies (30).To clarify these controversies, we solved the crystal structure of API-A in complex with two molecules of bovine trypsin. To the best of our knowledge, this is the first report on the three-dimensional structure of the double-headed Kunitz-type trypsin inhibitor in complex with two molecules of protease. On the basis of this structure, the two P1 residues have now been identified as Leu⁸⁷ and Lys¹⁴⁵ for reactive site 1 (RS1) and 2 (RS2), respectively. The results were further confirmed by site-directed mutagenesis. It was earlier shown that the first P1 residue Leu⁸⁷ interacts preferentially with chymotrypsin (30). In our structure, Leu⁸⁷ is snugly embedded in the S1 pocket of trypsin, as a consequence of the broad interface contributed by the surrounding residues. Comprehensive analyses of the two reactive site interfaces have provided functional insights into the novel inhibitory patterns of this unique double-headed protease inhibitor.     

Heme Regulatory Motifs in Heme Oxygenase-2 Form a Thiol/Disulfide Redox Switch That Responds to the Cellular Redox State

Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156–21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be −200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60–70% reduced, whereas oxidative stress conditions convert most (86–89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81–87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.Heme oxygenase (HO³ ; EC 1.14.99.3) catalyzes the O₂- and NADPH-dependent conversion of heme to biliverdin, carbon monoxide (CO), and iron in a reaction that is coupled to cytochrome P450 reductase. Then, biliverdin reductase catalyzes the NADPH-dependent reduction of biliverdin to the antioxidant bilirubin. Several recent reviews on HO (1–5) and biliverdin reductase (6) are available. HO is present in organisms from bacteria to eukaryotes and, as the only known enzyme that can degrade heme, plays a critical role in heme and iron homeostasis.There are two major HO isoforms in mammals: inducible HO-1, which is ancient and widely distributed among organisms from bacteria to man, and constitutively expressed HO-2, which emerged 250 million years ago with the amniotes (7). HO-1 is found in most tissues and is highly expressed in spleen and liver (8). Conversely, HO-2 has a narrow tissue distribution, exhibiting high expression levels in the brain, testes, and carotid body (8, 9). Both HO-1 and HO-2 catalyze the NADPH- and cytochrome P450 reductase-dependent degradation of heme to CO, iron, and biliverdin, which is quickly reduced to bilirubin in the presence of biliverdin reductase (10). Controlling cellular heme concentrations is crucial because heme is required as a prosthetic group by regulatory and redox proteins, yet concentrations higher than 1 μm free heme are toxic (11). Thus, as the only mammalian proteins known to degrade heme, HOs play a key role in cellular heme homeostasis; furthermore, in vitro and in vivo studies of cellular and tissue injuries, such as oxidative stress and hemin-induced cytotoxicity, indicate that HO is cytoprotective (9).HO-1 and HO-2 share high sequence and three-dimensional structural homology in their core domains (12, 13); however, their sequences diverge near their C termini, in which HO-2 contains two conserved heme regulatory motifs (HRMs), involving Cys²⁶⁵ in HRM1 and Cys²⁸² in HRM2⁴ (12, 14) (Fig. 1). It was shown recently that the HRMs in HO-2 do not bind heme per se but instead form a reversible thiol/disulfide redox switch that indirectly regulates the affinity of HO-2 for heme (14). However, for this redox switch to have any physiological consequence, the midpoint redox potential of the thiol/disulfide couple must be near the ambient intracellular redox potential, estimated to range from −170 to −250 mV (15).Open in a separate windowFIGURE 1.Major structural regions in HO-1 and HO-2. His²⁵ in HO-1 or His⁴⁵ in HO-2 is the heme-binding ligand.The HRM has been proposed to constitute a heme-binding site (16, 17) that regulates key metabolic processes from bacteria to humans. The HRM consists of a conserved Cys-Pro core sequence that is usually flanked at the N terminus by basic amino acids and at the C terminus by a hydrophobic residue. HRM/heme interactions have been proposed to regulate the activity and/or stability of proteins that play central roles in respiration and oxidative damage (18, 19), coordination of protein synthesis and heme availability in reticulocytes (20, 21), and controlling iron and heme homeostasis (22–26). An important component of the last process is HO-2.Here, we demonstrate that the C-terminal HRMs, which form a thiol/disulfide redox switch between Cys²⁶⁵ and Cys²⁸², exhibit a redox potential that falls well within the ambient cellular redox potential. By expressing HO-2 in bacterial and human cells and trapping the thiols using the isotope-coded affinity tag (ICAT) technique, it was shown that the redox state of the C-terminal HRMs in growing cells responds to the cellular redox state. The disulfide state is favored under oxidative conditions, and the dithiol state is predominant under reducing conditions. Thus, the HRMs act as a molecular rheostat that responds to the ambient intracellular redox potential and, based on earlier studies (14), controls activity of HO-2 by regulating heme binding to the enzyme.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Structural Basis of the Catalytic Mechanism Operating in Open-Closed Conformers of Lipocalin Type Prostaglandin D Synthase

Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD₂)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-β chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the β-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys⁶⁵ residue and its network of hydrogen bonds with the polar residues Ser⁴⁵, Thr⁶⁷, and Ser⁸¹, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys⁶⁵ residue is activated by its interaction with Ser⁴⁵ and Thr⁶⁷ and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.Prostaglandin (PG)⁶ D synthase (PGDS; PGH₂ d-isomerase (EC 5.3.99.2)) (1, 2) produces PGD₂, having 9α-hydroxy and 11-keto groups, from PGH₂, which bears the chemically labile 9,11-endoperoxide group and is produced as a common intermediate of all prostanoids by the action of cyclooxygenase (PGH₂ synthase). Two distinct types of PGDS have evolved from phylogenetically distinct protein families (2, 3). One is hematopoietic PGDS (H-PGDS), which belongs to the σ class of GSH S-transferases (4, 5), and the other is lipocalin type PGDS (L-PGDS), a member of the lipocalin family (6, 7). L-PGDS is the only enzyme in the lipocalin family and is identical to β-trace, a major protein in human cerebrospinal fluid (8, 9). Although H-PGDS and L-PGDS catalyze the same reaction, their amino acid sequences and tertiary structures are quite different from each other, indicating that these enzymes are a new example of functional convergence (2, 3).L-PGDS is expressed in the heart, central nervous system, and male genital organs of various mammals and is involved in various physiological and pathological functions (reviewed in Refs. 6 and 7). In the brain, L-PGDS produces PGD₂, which is involved in the regulation of pain and non-rapid eye movement sleep, as was shown in studies using gene knock-out mice (10, 11) and human enzyme transgenic mice (12). L-PGDS is regulated by SOX9 and is involved in the differentiation of male genital organs (13–15). This enzyme is also expressed in adipocytes (16), vascular smooth muscle cells (17), and myocardial cells (18, 19) and is involved in adipocyte differentiation, the progression of arteriosclerosis (20), and the protection against hypoxemia (18) or ischemia/reperfusion injury (19). L-PGDS binds various lipophilic compounds, such as retinoids (21), bilirubin, biliverdin (22), gangliosides (23), and amyloid-β peptides (24, 25), with high affinity, acting as an extracellular transporter of these compounds and serving as an endogenous amyloid-β chaperone to prevent amyloid deposition in vivo (24).Although many biochemical and physiological studies suggest important roles of PGD₂ and L-PGDS/β-trace in the regulation of sleep and other biological functions, the crystal structure of L-PGDS has not been resolved. In this study, we determined the crystal structures of two different forms of the Δ^1–24-C65A mutant of mouse L-PGDS in both open and closed conformations. L-PGDS was shown to possess a typical lipocalin fold, the β-barrel, which is a unique structural component specific to L-PGDS and comprises a mobile E-F loop and a large central cavity with two compartments. By performing site-directed mutagenesis of Δ^1–24-L-PGDS and the Δ^1–24-C65A mutant, we found that the Cys⁶⁵ surrounded by the hydroxyl side chains of Ser⁴⁵, Thr⁶⁷, and Ser⁸¹ was activated to contribute to the catalysis by L-PGDS.     

Binding Modes of Aromatic Ligands to Mammalian Heme Peroxidases with Associated Functional Implications: CRYSTAL STRUCTURES OF LACTOPEROXIDASE COMPLEXES WITH ACETYLSALICYLIC ACID, SALICYLHYDROXAMIC ACID, AND BENZYLHYDROXAMIC ACID*

The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA) show that all the three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group of their hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen-bonds, one each with heme iron, His-109 N^ϵ2, and Gln-105 N^ϵ2. In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly with Fe-OH distances of 3.0 and 3.2Å respectively. The OH is also hydrogen bonded to His-109 N^ϵ2 and Gln-105N^ϵ2. The plane of benzene ring of ASA is inclined at 70.7° from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane with inclinations of 15.7 and 6.2°, respectively. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding.Lactoperoxidase (LPO)⁴ (EC 1.11.1.7) is a member of the family of glycosylated mammalian heme-containing peroxidase enzymes which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase. These enzymes also show functional similarities to non-homologous plant and fungal peroxidases because they follow a similar scheme of reaction (1–3), but their modes of ligand binding differ considerably. Furthermore, the association of the prosthetic heme group in mammalian peroxidases is through covalent bonds (4–9), whereas covalent linkages are absent in other peroxidases (10–14). Among the four mammalian peroxidases, the prosthetic heme group is linked through three covalent bonds in MPO, whereas in LPO, EPO, and thyroid peroxidase only two covalent linkages are formed. So far the detailed crystal structures of only two mammalian peroxidases, MPO and LPO, are known (15–20). One of the most striking differences between these two mammalian peroxidases is concerned with the basic structural organization in which MPO exists as a covalently linked dimer, whereas LPO is a monomeric protein. At present the fundamental questions pertaining to mammalian heme peroxidases are (i) what distinguishes between the aromatic ligands that one ligand acts as a substrate, whereas the other ligand works as an inhibitor and (ii) how the substrate and inhibitor specificities differ between two enzymes lactoperoxidase and myeloperoxidase.Lactoperoxidase oxidizes inorganic ions, preferentially thiocyanate (SCN⁻), and to a lesser extent, bromide (Br⁻), whereas in the case of myeloperoxidase the chloride (Cl⁻) ion is a preferred substrate (21, 22). The mammalian peroxidases including LPO are also involved in catalyzing the single electron oxidation of various physiologically important organic aromatic substrates including phenols (23, 24), catecholamines, and catechols (25–27) as well as other experimental model compounds such as aromatic amines (28), polychlorinated biphenyls (29), steroid hormones (30–32), and polycyclic aromatic hydrocarbons (33). However, the mode of binding of aromatic ligands and associated functional implications are not yet clearly understood. Surprisingly, the structural data on the complexes of mammalian peroxidases with aromatic ligands are conspicuously lacking. The only available structural report is on the complex of MPO with salicylhydroxamic acid (SHA) (34). Even in this case, the coordinates of this structure are not available for a detailed analysis. In the case of non-homologous plant peroxidases, a few crystal structures of their complexes with aromatic compounds are available (35–38), but their modes of binding are not very similar to those of mammalian peroxidases because the distal ligand binding sites in mammalian and plant peroxidases differ markedly. In this regard it is pertinent to note that the substrate binding site in peroxidases, in general, is observed at the δ-heme edge of the heme moiety on the distal side; in plant peroxidases an additional ligand binding site has also been observed at γ-heme edge (39–41). Unlike those in mammalian peroxidases where the heme moiety is buried deeply in the protein core, in plant peroxidases it is located close to the surface of the protein. Therefore, to characterize the mode of binding of the aromatic substrates and aromatic inhibitors and also for defining the subsites in the substrate binding site, we have determined the crystal structures of three complexes of bovine lactoperoxidase with aromatic ligands, acetylsalicylic acid (ASA), SHA, and benzylhydroxamic acid (BHA). Acetylsalicylic acid can be oxidized by lactoperoxidase to ASA free radical (42), whereas both salicylhydroxamic acid and benzylhydroxamic acid act as potent inhibitors of mammalian peroxidases (43–47). The determination of binding characteristics of these compounds having different actions has helped in establishing the relationship between the modes of binding and their potential actions as the substrates and inhibitors. To the best of our knowledge, this is the first structural report on the modes of binding of three aromatic ligands, ASA, SHA, and BHA, to LPO as well as the first structural study of the complexes of any mammalian peroxidase with ASA and BHA. These studies have shown that ASA, SHA, and BHA bind to LPO at the substrate binding site on the distal side. The SHA and BHA directly interact with the heme iron, whereas ASA interacts through the heme water molecule, which in turn is hydrogen-bonded to the heme iron. These studies have provided a greater insight into the mechanisms of substrate and inhibitor binding in the two mammalian peroxidases.     

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2–4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9–14, 72).The sortase A (SrtA)⁷ enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between the threonine and glycine residues, forming a thioacyl-linked protein-sortase intermediate (16). It then catalyzes the formation of an amide bond between the carboxyl group of the threonine and the cell wall precursor molecule lipid II (undecaprenyl-pyrophosphate-MurNAc(-l-Ala-d-iGln-l-Lys(NH₂-Gly₅)-d-Ala-d-Ala)-β1–4-GlcNAc)), creating a protein-lipid II-linked product that is incorporated into the peptidylglycan via the transglycosylation and transpeptidation reactions of bacterial cell wall synthesis (18–20). Over 900 sortase-attached proteins in 72 different strains of bacteria have thus far been identified (21, 22). The vast majority of these proteins contain a COOH-terminal sorting signal harboring an LPXTG motif and are anchored to the cell wall by enzymes closely related to SrtA.In vitro studies of SrtA have begun to define the mechanism of transpeptidation. SrtA consists of two parts: an unstructured amino-terminal tail that contains a stretch of nonpolar residues that embed it in the membrane and an autonomously folded catalytic domain that competently performs the transpeptidation reaction in vitro (SrtA_ΔN59, residues 60–206) (16, 23–25). Catalysis occurs through a ping-pong mechanism that is initiated when the thiol group of amino acid Cys¹⁸⁴ nucleophilically attacks the carbonyl carbon of the threonine residue within the LPXTG sorting signal (16, 23–25). This forms a transient tetrahedral intermediate that, upon breakage of the threonine-glycine peptide bond, rearranges into a more stable thioacyl enzyme-substrate linkage. SrtA then joins the terminal amine group within the pentaglycine branch of lipid II to the carbonyl carbon of the threonine, creating a second tetrahedral intermediate that is resolved into the lipid II-linked protein product (23).Sortase enzymes contain three conserved residues within their active sites: His¹²⁰, Cys¹⁸⁴, and Arg¹⁹⁷ (SrtA numbering). These residues play a critical role in catalysis, since their mutation in SrtA causes severe reductions in enzyme activity (16, 26–30). Although it is well established that Cys¹⁸⁴ forms a covalent linkage to the sorting signal, the functions of His¹²⁰ and Arg¹⁹⁷ are controversial. A variety of disparate functions have been ascribed to Arg¹⁹⁷. These include deprotonating Cys¹⁸⁴ (28), deprotonating lipid II (31), or stabilizing the binding of either the LPXTG sorting signal (28, 32) or oxyanion intermediates (31, 32). Different functions have also been proposed for His¹²⁰. Originally, it was suggested that it activated Cys¹⁸⁴ by forming an imidazolium-thiolate ion pair (26). However, subsequent pK_a measurements revealed that both His¹²⁰ and Cys¹⁸⁴ are predominantly uncharged at physiological pH values, leading to the suggestion that His¹²⁰ functions as a general base during catalysis (33). Most recently, it has been proposed that the most active form of the enzyme contains His¹²⁰ and Cys¹⁸⁴ in their charged states but that only a small fraction of SrtA exists in this form (∼0.06%) prior to binding to the sorting signal (25).NMR and crystal structures of SrtA_ΔN59 have revealed that it adopts an eight-stranded β-barrel fold (31, 34). Other sortase enzymes have also been shown to possess a similar overall structure, including SrtB from S. aureus (27, 35), SrtB from Bacillus anthracis (27, 36), SrtA from S. pyogenes (37), and the SrtC-1 and SrtC-3 enzymes from S. pneumoniae (38). However, the molecular basis of substrate recognition remains poorly understood, because all of the structures reported to date have not contained a sorting signal bound to the enzyme. The lone exception is the crystal structure of SrtA_ΔN59 bound to an LPETG peptide (31). However, in this structure the peptide substrate is bound nonspecifically (see below) (32, 39).In this paper, we report the structure and dynamics of SrtA covalently bound to an analog of the LPXTG sorting signal. The structure of the complex resembles the thioacyl intermediate of catalysis, providing insights into the molecular basis of binding of the LPXTG sorting signal and the functions of key active site residues. Notably, the mechanism of substrate binding visualized in the NMR structure differs substantially from a previously reported crystal structure of SrtA_ΔN59 non-covalently bound to a LPETG peptide (31). We have also used NMR chemical shift mapping experiments to localize the binding site for a triglycine cell wall substrate analog. A mechanism of transpeptidation compatible with these new data is proposed.     

Myosin II Tailpiece Determines Its Paracrystal Structure, Filament Assembly Properties, and Cellular Localization

Non muscle myosin II (NMII) is a major motor protein present in all cell types. The three known vertebrate NMII isoforms share high sequence homology but play different cellular roles. The main difference in sequence resides in the C-terminal non-helical tailpiece (tailpiece). In this study we demonstrate that the tailpiece is crucial for proper filament size, overcoming the intrinsic properties of the coiled-coil rod. Furthermore, we show that the tailpiece by itself determines the NMII filament structure in an isoform-specific manner, thus providing a possible mechanism by which each NMII isoform carries out its unique cellular functions. We further show that the tailpiece determines the cellular localization of NMII-A and NMII-B and is important for NMII-C role in focal adhesion complexes. We mapped NMII-C sites phosphorylated by protein kinase C and casein kinase II and showed that these phosphorylations affect its solubility properties and cellular localization. Thus phosphorylation fine-tunes the tailpiece effects on the coiled-coil rod, enabling dynamic regulation of NMII-C assembly. We thus show that the small tailpiece of NMII is a distinct domain playing a role in isoform-specific filament assembly and cellular functions.Non muscle myosin II (NMII)² is a major motor protein present in all cell types participating in crucial processes, including cytokinesis, surface attachment, and cell movement (1–3). NMII units are hexamers of two long heavy chains with two pairs of light chains attached. NMII heavy chain is composed of a globular head containing the actin binding and force generating ATPase domains, followed by a large coiled-coil rod that terminates with a short non-helical tailpiece (tailpiece). To carry out its cellular functions, NMII assembles into dimers and higher order filaments by interactions of the coiled-coil rod (4). The assembly process is governed by electrostatic interactions between adjacent coiled-coil rods containing alternating charged regions with specific periodicity (5–9) and is enhanced by activation of the motor domain through regulatory light chain phosphorylation (10–12). The charge periodicity also determines the register and orientation of each NMII hexamer in the filament. Additionally the C-terminal region of the coiled-coil rod contains a distinctive positively charged region and the assembly-competence domains that are crucial for proper filament assembly (5–9, 13).Three isoforms of NMII (termed NMII-A, NMII-B, and NMII-C) have been identified in mammals (14–16). Although NMII isoforms share somewhat overlapping roles, each isoform has distinctive tissue distribution and specific functions. NMII-A is important for neural growth cone retraction (17, 18) and is distributed to the front of migrating endothelial cells (19). While NMII-B participates in growth cone advancement (20) and was detected in the retracting tails of migrating endothelial cells (19). Furthermore NMII-A and NMII-B have an opposing effect on motility, since depletion of NMII-A leads to increased motility while NMII-B depletion hinders motility (21, 22). NMII-C plays a role in cytokinesis (23) and has distinct distribution in neuronal cells (24). Furthermore one NMII isoform only partly rescue cells in which siRNA was used to reduce the expression of another isoform (23, 25). This functional diversity is achieved despite a significant amino acid sequence identity between the isoforms (overall 64–80%), and the origin of these differential distributions and functions is not completely understood.Recent studies suggest that the C-terminal portion of NMII-A and NMII-B, particularly the last ∼170 amino acids, is responsible for the differential distribution of these NMII isoforms (26, 27). It was shown that swapping this region between NMII-A and NMII-B resulted in chimeric proteins, which adopted cellular localization according to the C-terminal part (26). This C-terminal ∼170 amino acid coiled-coil region contains the assembly-competence domains and other regions that are critical for filament assembly (5–9, 13) as well as the non-helical tailpiece. As the small tailpiece is also an important regulator of NMII filament assembly (27, 28) capable of changing NMII filament assembly properties; and phosphorylation of NMII tailpiece was shown to interfere with filament assembly (29–33) the tailpiece may be important for allowing NMII to perform its dynamic tasks. Because the coiled-coil regions are highly conserved between NMII isoforms, while the tailpiece is the most divergent, it is therefore a good candidate for mediating NMII isoform-specific functions. However, the exact mechanism by which the tailpiece affects NMII function is not fully understood. Here we show that the tailpiece serves as an isoform-specific control mechanism modulating filament order, assembly, and cellular function.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.