Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity

Lactobacillus reuteri mucus-binding protein (MUB) is a cell-surface protein that is involved in bacterial interaction with mucus and colonization of the digestive tract. The 353-kDa mature protein is representative of a broadly important class of adhesins that have remained relatively poorly characterized due to their large size and highly modular nature. MUB contains two different types of repeats (Mub1 and Mub2) present in six and eight copies, respectively, and shown to be responsible for the adherence to intestinal mucus. Here we report the 1.8-Å resolution crystal structure of a type 2 Mub repeat (184 amino acids) comprising two structurally related domains resembling the functional repeat found in a family of immunoglobulin (Ig)-binding proteins. The N-terminal domain bears striking structural similarity to the repeat unit of Protein L (PpL) from Peptostreptococcus magnus, suggesting binding in a non-immune Fab-dependent manner. A distorted PpL-like fold is also seen in the C-terminal domain. As with PpL, Mub repeats were able to interact in vitro with a large repertoire of mammalian Igs, including secretory IgA. This hitherto undetected activity is consistent with the current model that antibody responses against commensal flora are of broad specificity and low affinity.     

The X-ray Crystal Structure of Human Aminopeptidase N Reveals a Novel Dimer and the Basis for Peptide Processing

Human aminopeptidase N (hAPN/hCD13) is a dimeric membrane protein and a member of the M1 family of zinc metallopeptidases. Within the rennin-angiotensin system, its enzymatic activity is responsible for processing peptide hormones angiotensin III and IV. In addition, hAPN is also involved in cell adhesion, endocytosis, and signal transduction and it is an important target for cancer therapy. Reported here are the high resolution x-ray crystal structures of the dimeric ectodomain of hAPN and its complexes with angiotensin IV and the peptidomimetic inhibitors, amastatin and bestatin. Each monomer of the dimer is found in what has been termed the closed form in other M1 enzymes and each monomer is characterized by an internal cavity surrounding the catalytic site as well as a unique substrate/inhibitor-dependent loop ordering, which in the case of the bestatin complex suggests a new route to inhibitor design. The hAPN structure provides the first example of a dimeric M1 family member and the observed structural features, in conjunction with a model for the open form, provide novel insights into the mechanism of peptide processing and signal transduction.     

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.     

Crystal Structure of Methanocaldococcus jannaschii Trm4 Complexed with Sinefungin

tRNA:m⁵C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 Å and 2.3 Å resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix α8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.     

Crystal Structure of a Left-Handed Z-DNA Hexamer,d(CG)3, Duplex Complexed with Synthetic Polyamine Reveals Binding of a Polyamine in the Minor Groove

Abstract

As a series of X-ray structural studies of Z-DNA Polyamine complex, the crystal structure of Z-DNA hexamer, d(CG)₃, duplex complexed with a synthetic polyamine, N,N′-bis(2-aminoethyl)-1,2-ethanediamine, NH₂-(CH₂)₂-NH-(CH₂)₂-NH-(CH₂)₂-NH₂ [PA(222)], has been determined.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.     

Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7

Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.     

Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif

The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase α is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.     

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C^pro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C^pro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C^pro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3C^pro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4′ region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C^pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C^pro.     

Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin β-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I)₂GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.Many transmembrane signaling systems consist of specific G protein-coupled receptors (GPCRs)³ that transduce a diverse array of extracellular stimuli into intracellular signaling events (1). GPCRs modulate the activity of numerous effector molecules and regulate multiple biological functions including neurotransmission, sensory perception, cardiovascular function, development, and cell growth and differentiation (2). To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and duration, these signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation; 1) desensitization, in which a receptor becomes refractory to continued stimuli; 2) endocytosis, where receptors are removed from the cell surface; 3) down-regulation, where total receptor levels are decreased (3, 4). Agonist-dependent regulation is primarily mediated by GPCR kinases that specifically phosphorylate activated GPCRs and initiate the recruitment of arrestins. Arrestins are divided into two major classes, visual and non-visual, based on their localization and function. The non-visual arrestins, arrestin2 and 3 (also termed β-arrestin1 and -2, respectively), are broadly distributed and function in multiple processes including GPCR desensitization, trafficking, and signaling (4–6).Initial structural insight on arrestins was provided by the x-ray crystal structure of bovine arrestin1 (7, 8), whereas the crystal structures of C-terminal-truncated (9) and wild type (10) bovine arrestin2 and salamander arrestin4 (11) have also been solved. In general, arrestins are composed of two major domains made up of β strands and connecting loops that are held together by a polar core region consisting of buried salt bridges. It has been proposed that arrestins adopt an active conformation upon binding to phosphorylated receptors, which disrupts the polar core resulting in the release of the C-terminal tail (12). Disruption of the polar core by point mutation of Arg-169 generates a constitutively active arrestin2, which mimics the active state. This mutated arrestin binds to the β₂-adrenergic receptor (β₂AR) in a phosphorylation-independent manner, induces internalization of a δ-opioid receptor lacking phosphorylation sites (13), and has increased binding to clathrin and AP-2 (14).A role for non-visual arrestins in GPCR endocytosis was first described for the β₂AR (15, 16), although it is now evident that arrestins regulate the trafficking of multiple GPCRs as well as additional classes of receptors (4). An early step in this process involves arrestin binding to an activated phosphorylated receptor that enhances arrestin interaction with the endocytic proteins, clathrin, and AP-2 (16, 17). An additional important step in this process involves arrestin interaction with phosphoinositides such as phosphatidylinositol diphosphate and trisphosphate (18). Although the dynamics of these interactions have not been studied, arrestin2 and -3 have been shown to interact specifically and stoichiometrically with clathrin (16). Furthermore, fluorescence microscopy reveals that activated β₂AR, arrestin2, clathrin, and AP-2 all colocalize upon receptor stimulation (16). The primary clathrin binding determinant in arrestin2, LIELD, spans residues 376–380 and is located in an extended disordered loop that immediately precedes the final C-terminal β-strand (10, 19). This region, the clathrin binding box, is consistent with a consensus motif, LϕXϕ(D/E) (where ϕ is a bulky hydrophobic residue, and X represents any polar amino acid), established in other clathrin-binding proteins including AP-2 (20), AP180 (21), amphiphysin (22), and epsin (23). Importantly, the mutation of this motif in arrestin3 and its deletion in arrestin2 significantly disrupts clathrin binding and receptor endocytosis (14, 19). A mutagenesis study of clathrin localized an arrestin binding site to the N-terminal domain of the clathrin heavy chain, specifically residues Glu-89, Lys-96, and Lys-98 (24). Moreover, a crystal structure of clathrin-(1–363) in complex with an arrestin3 peptide (residues 369–381) supports the mutagenesis data and the predicted location of the arrestin-clathrin interaction site (25).To further elucidate the mechanisms involved in mediating arrestin/clathrin interaction, we have determined the crystal structure of clathrin with the short (arrestin2S) and long (arrestin2L) isoforms of arrestin2, which differ by an 8-amino acid insert between β strands 18 and 19 (26). Our results identify an additional and unique interaction encoded in the arrestin2L isoform that is distinct from the previously well characterized interaction involving the LϕXϕ(D/E) motif. Specifically, we observe that the 8 amino acid splice loop in arrestin2L interacts with a pocket formed by blades 4 and 5 in clathrin. Biochemical and cell biological analysis confirm a role for both binding sites in arrestin2L/clathrin interaction and demonstrate an essential role of these interactions in arrestin-mediated GPCR endocytosis.     

Crystal Structure of the Human Fatty Acid Synthase Enoyl-Acyl Carrier Protein-Reductase Domain Complexed with Triclosan Reveals Allosteric Protein-Protein Interface Inhibition

Human fatty acid synthase (FAS) is a large, multidomain protein that synthesizes long chain fatty acids. Because these fatty acids are primarily provided by diet, FAS is normally expressed at low levels; however, it is highly up-regulated in many cancers. Human enoyl-acyl carrier protein-reductase (hER) is one of the FAS catalytic domains, and its inhibition by drugs like triclosan (TCL) can increase cytotoxicity and decrease drug resistance in cancer cells. We have determined the structure of hER in the presence and absence of TCL. TCL was not bound in the active site, as predicted, but rather at the protein-protein interface (PPI). TCL binding induces a dimer orientation change that causes downstream structural rearrangement in critical active site residues. Kinetics studies indicate that TCL is capable of inhibiting the isolated hER domain with an IC₅₀ of ∼55 μm. Given the hER-TCL structure and the inhibition observed in the hER domain, it seems likely that TCL is observed in the physiologically relevant binding site and that it acts as an allosteric PPI inhibitor. TCL may be a viable scaffold for the development of anti-cancer PPI FAS inhibitors.     

Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.