Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase ��-Domain and Kringle 5 of the Substrate

Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK·Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK·Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys⁴¹⁴. Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys⁴¹⁴ with Ala substitutions of charged residues in the SK β-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys²⁵⁶/Lys²⁵⁷ double mutant. Binding of bovine Pg (BPg) to the SK·Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK·labeled Pm·BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys²⁵⁶/Lys²⁵⁷ mutant. BPg was activated by SK·Pm with a K_m indistinguishable from the K_D for BPg binding to form the ternary complex, whereas the SK Lys²⁵⁶/Lys²⁵⁷ mutant did not support BPg activation. We conclude that SK residues Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK·kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.Streptokinase (SK)⁶ activates the human fibrinolytic system by activating plasminogen (Pg) through a unique mechanism that is responsible for the use of SK as a thrombolytic drug and its role as a key pathogenicity factor in Group A streptococcal infection (1, 2). The crystal structure of SK bound to the catalytic domain of plasmin (μPm) shows that SK consists of three β-grasp, tightly folded domains, α, β, and γ, linked by flexible segments (3). In solution, SK is highly flexible and behaves hydrodynamically like three beads on a string (4). When bound to μPm, SK assumes a highly ordered structure resembling a three-sided crater surrounding the catalytic site that provides an exosite(s) for binding the catalytic domain of Pg as a substrate (3, 5). In the first step of the SK-mediated Pg activation pathway, SK binds the catalytic domain of the Pg zymogen in a rapid equilibrium process and inserts its NH₂-terminal Ile¹ residue into the NH₂-terminal binding cleft of Pg, activating the catalytic site nonproteolytically (6–10). Although structural proof is lacking, SK Ile¹ presumably forms a critical salt bridge with Asp^740(194) (plasminogen numbering; chymotrypsinogen numbering is in parentheses) that initiates conformational activation of the substrate binding site and oxyanion hole required for proteolytic activity (6, 8–10). The activated SK·Pg* complex binds a second molecule of Pg as a specific substrate and cleaves it at Arg^561(15)-Val^562(16) to form the fibrin-degrading proteinase, plasmin (Pm) (10–14). Proteolytic generation of Pm is propagated by formation of a high affinity SK·Pm complex that converts the remaining free Pg into Pm (5, 11).[Glu]Pg, the full-length form of Pg circulating in blood, consists of an NH₂-terminal PAN (Pg/Apple/Nematode (15, 16)) module, followed by five kringle domains (K1–K5), and the trypsin-like serine proteinase catalytic domain (17). Formation of the SK·Pg* and SK·Pm catalytic complexes and Pg substrate binding are inhibited by the lysine analog, 6-aminohexanoic acid (6-AHA), which binds to lysine-binding sites (LBS) located primarily in kringles K1, K4, and K5 of Pg and Pm (10, 11, 18–23). Cleavage of the Lys⁷⁷-Lys⁷⁸ peptide bond in [Glu]Pg by Pm releases the PAN module and generates the truncated form, [Lys]Pg. Formation of [Lys]Pg is accompanied by a conformational change of [Glu]Pg from a compact, closed α-conformation to a partially extended β-conformation with expression of higher affinity LBS for 6-AHA (24, 25). The fourth kringle module mediates a second conformational change, from the β-conformation to the extended γ-conformation (25).Binding of SK to [Glu]Pg is independent of LBS, with a dissociation constant of 100–150 nm, whereas formation of SK·[Lys]Pg is LBS-dependent with a 13–20-fold higher affinity that is reduced to that of [Glu]Pg by saturating concentrations of 6-AHA (10, 21). Activation of the catalytic domain in [Lys]Pm increases affinity for SK about 830-fold, which is reduced 11–20-fold by 6-AHA (5, 21). Interaction of the COOH-terminal Lys⁴¹⁴ residue of SK with a Pg/Pm kringle domain is responsible for the LBS-dependent enhancement of the affinity of SK·[Lys]Pg* and SK·Pm catalytic complex formation (22). Recent rapid reaction kinetic studies of the SK·Pm binding pathway demonstrated that interaction of Lys⁴¹⁴ with a Pm kringle enhances formation of an initial rapid equilibrium SK·Pm encounter complex, succeeded by two sequential, tightening conformational changes, to achieve an overall dissociation constant of ∼12 pm (26). The Pg/Pm kringle domain responsible for the enhancement of SK·Pg* and SK·Pm complex formation is not known. Productive interaction of Pg as a substrate of the SK·Pg*/Pm complexes is also greatly inhibited by saturating 6-AHA (11). Kinetic and equilibrium binding studies of SK-mediated Pm formation resolved the conformational activation process from the coupled proteolytic generation of Pm (10, 11). The kinetic approach demonstrated that Lys⁴¹⁴ deletion reduced the affinity of formation of the SK·Pg* catalytic complex specifically, whereas the subsequent LBS-dependent proteolytic formation of Pm was unaffected, indicating that Pg substrate recognition is mediated by a structurally distinct region of SK and an unknown kringle (22).Previous structure-function studies have yielded diverse interpretations and conclusions regarding the structural basis of LBS-dependent Pg substrate recognition (23, 27–34). Each of the three domains of SK has been implicated in this regard (29, 30, 35, 36), and binding of two Pg molecules to the residue 1–59 sequence of the α-domain has been reported (36). In particular, segments 16–36, 41–48, 48–59, and 88–97 of the SK α-domain have been concluded to play a role in Pg substrate recognition (32, 33, 37, 38). For several SK mutants, a complex mixture of functional effects on their binding to [Glu]Pg and its conformational and proteolytic activation has been reported (28, 31, 33). Some of these effects may result from the inherent flexibility of SK when bound to Pg or Pm (39), and others may be due to the use of kinetic approaches that do not clearly discriminate between conformational and proteolytic activation.Some observations implicate a protruding hairpin loop called the 250-loop (residues Ala²⁵¹–Ile²⁶⁴) in the SK β-domain in Pg substrate recognition (27, 28, 31, 34). This loop is disordered in the structure of the SK·μPm complex but is ordered in the structure of the isolated β-domain (3, 40). Deletion of the 250-loop, Ala substitution of Lys²⁵⁶ and Lys²⁵⁷ at the apex of the loop, and substitution of multiple residues near and within the loop resulted in disparate effects on K_m and k_cat for [Glu]Pg activation (27, 28, 31). The conclusions of these studies were that Lys²⁵⁶ and Lys²⁵⁷ are involved in SK binding and conformational activation of [Glu]Pg in addition to proteolytic processing of Pg as a substrate. Some of these studies are problematic because the natural NH₂-terminal Ile¹ residue necessary for conformational activation is preceded either by an additional methionine (27, 31) or maltose-binding protein (28) in the recombinant SK species used.Because of the diverse conclusions regarding the functional properties of the 250-loop mutations and the possibility of other potential Pg substrate binding sites, the present studies were undertaken to resolve the function of residues in the 250-loop in LBS-dependent Pg substrate recognition by the SK·Pg* complex. The kringle domain of Pg involved in Pg substrate recognition has not been clearly identified but has been suggested to be K5 (27) on the basis that the isolated β-domain bound Pg (30) and K5 (29) in an LBS-dependent manner. Given the general specificity of Pg kringles for COOH-terminal Lys residues and zwitterionic ligands, such as 6-AHA, and the internal sequence of the 250-loop, it appeared possible that a pseudolysine motif on SK was involved. In the binding of a 30-residue peptide from plasminogen binding Group A streptococcal M-like protein (PAM), VEK-30, to K2 of Pg, Castellino and co-workers (41, 42) showed by crystallography and mutagenesis that residues with cationic (Arg and His) and anionic side chains (Glu) arranged spatially on a helix constituted a pseudolysine structure similar to 6-AHA that binds specifically to the LBS of K2. Additional evidence for pseudolysine structures in Pg binding comes from studies of α-enolase from Streptococcus pneumoniae, which has a 9-residue internal binding site for Pg containing essential basic (two Lys residues) and acidic (Asp and Glu residues) located on a surface loop (43, 44).To determine whether a similar SK structure is involved in [Lys]Pg substrate recognition, anionic and cationic residues in the 250-loop were substituted with Ala and characterized in kinetic studies using methods that resolve conformational and proteolytic activation. Studies with [Lys]Pg and mini-Pg, which contains only K5 and the catalytic domain, showed that Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ facilitate LBS-dependent substrate recognition through interactions with K5. The absence of evidence for a pseudolysine structure in the 250-loop is compatible with the established atypical specificity of K5 for cationic ligands, such as benzamidine, N^α-acetyl-Lys-methyl ester, 6-aminohexane, and 5-aminopentane, in addition to zwitterionic ligands (19, 45–47). The studies resolve for the first time the structural features of SK that mediate the LBS-dependent interactions that enhance affinity of SK·Pg* and SK·Pm catalytic complex formation and those that facilitate binding of Pg as a substrate of these complexes.     

Studies on the DIDS-binding Site of Monocarboxylate Transporter 1 Suggest a Homology Model of the Open Conformation and a Plausible Translocation Cycle

Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys³⁸, Lys⁴⁵, Lys²⁸², and Lys⁴¹³. K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO₃⁻ groups interact with positively charged Lys²⁸² together with Lys⁴⁵ and/or Lys⁴¹³. This positions one DIDS isothiocyanate group close to Lys³⁸, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys³⁸ or Lys²⁹⁰ of MCT1 and Lys¹⁶⁰ or Lys¹⁶⁴ of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys³⁸ in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys³⁸ accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp³⁰²⁻ and Asp³⁰⁶⁺, an ion pair already identified as important for transport and located adjacent to Phe³⁶⁰, which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data.Monocarboxylate transporter 1 (MCT1)³ is a member of the monocarboxylate transporter family (SLC16) of which there are 14 known members encoded by both the human and mouse genomes (1). All of the members of this family are thought to have 12 transmembrane alpha helices (TMs) with a large loop between TMs 6 and 7 and the C and N termini facing the cytosol (2, 3). The only members of the MCT family that have been shown to catalyze transport of monocarboxylates such as l-lactate across the plasma membrane are isoforms 1–4 (4–8). This transport is proton-linked and leads to the net uptake or release of lactic acid from cells, which is critical for metabolic pathways such as anaerobic glycolysis, gluconeogenesis, and lactate oxidation (9). MCT8 is a high affinity thyroid hormone transporter (10), whereas MCT10 (TAT1) is an aromatic amino acid transporter (11). The other members of the MCT family remain to be characterized.MCT1 is the most widely distributed member of the MCT family and was first identified as the lactate transporter present in red blood cells where its kinetics and substrate and inhibitor specificity were investigated in detail (9, 11, 12). These studies revealed that MCT1 can be inhibited by stilbene disulfonate derivatives such as DIDS and 4,4′-dibenzamido-stilbene-2,2′-disulfonate (DBDS). DIDS was shown to exhibit a rapid reversible inhibition of transport that was competitive with respect to l-lactate. This is followed by a slowly developing irreversible inhibition that is not exhibited by DBDS and is thought to be caused by one of the isothiocyanate groups of DIDS attacking a lysine residue on MCT1 (13–15). Prolonged incubation with DIDS also led to a fraction of the MCT1 becoming cross-linked to a 70-kDa glycoprotein that was identified as embigin, also known as gp70 (16). Embigin has a short intracellular C terminus, a single TM sequence containing a glutamic acid residue, and a large extracellular N terminus containing two immunoglobulin domains (17, 18). Subsequent studies revealed that either embigin or, more frequently, the homologous protein basigin (also known as CD147) is required as a chaperone to take MCT1 to the membrane (19) where the two proteins must remain associated for transport activity to be maintained (20, 21).Expression of MCTs 1, 2, and 4 in Xenopus laevis oocytes has enabled their further characterization and the effects of site-directed mutagenesis to be investigated (4, 5, 7, 8, 22–24). Such studies, together with homology modeling have enabled us to propose a three-dimensional structure of MCT1 based around the published structure of the Escherichia coli glycerol-3-phosphate transporter (Protein Data Bank 1PW4) (24). This model can account for the effects of mutating a range of amino acids, including some that disrupt the interaction with basigin, and has led to the proposal that the single TM of basigin or embigin lies between TMs 3 and 6 of MCT1. The model also reveals exofacial lysines that are present in MCT1 that might be responsible for the irreversible inhibition of MCT1 by DIDS and the cross-linking of MCT1 to embigin. In rat MCT1 these residues are Lys³⁸, Lys⁴⁵, Lys²⁸², Lys²⁸⁴, Lys²⁹⁰, and Lys⁴¹³. In this paper, we use site-directed mutagenesis of these lysine residues to identify which of them are involved in DIDS binding to MCT1. In addition we use site-directed mutagenesis of embigin to demonstrate that Lys¹⁶⁰ and Lys¹⁶⁴ are involved in its cross-linking to MCT1. Our new data allow us to propose a modified structural model of MCT1 in its outward facing conformation that binds DIDS. This model is consistent with the site-directed mutagenesis data and also suggests a mechanism for the translocation cycle that involves Lys³⁸ as well as Asp³⁰² and Arg³⁰⁶ that have already been identified as important for transport (23, 24). We have also been able to model a structure of MCT1 bound to embigin that is consistent with published data.     

Increased Protein Stability of FGF1 Can Compensate for Its Reduced Affinity for Heparin

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1·fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1·FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.FGF1 (fibroblast growth factor 1) belongs to a family of polypeptide growth factors comprising in humans 22 structurally related proteins (1, 2). The signaling induced by the growth factor leads to a wide range of cellular responses during development as well as in adult life, such as growth regulation, differentiation, survival, stress response, migration, and proliferation of different cell types (3). The biological activity of FGF1 is exerted through binding to four high affinity cell surface receptors (FGFR1–4), resulting in receptor dimerization and transphosphorylation in its tyrosine kinase domain (4, 5). The activated FGFR³ induces cellular response by initiating several signaling cascades, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase/Akt, and phospholipase C-γ (PLC-γ) pathways (6).In addition to FGFRs, FGF1 binds to heparan sulfates (HS) associated with proteoglycans at the cell surface and in the extracellular matrix (7). Among the physiological sugars, the highest affinity for FGF1 is shown by heparin, a widely used linear, highly sulfated polysaccharide composed of 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine units (8).Despite many years of research, there is still controversy regarding the molecular role of heparin/HS in FGF1- and FGF2-induced signaling. Thus, the question of whether or not the linkage of two molecules of the growth factor by heparin/HS is an absolute prerequisite for induction of FGFR dimerization is still open. Numerous studies have concluded that the presence of heparin/HS is obligatory for FGF signaling. It is widely believed that heparin/HS is directly involved in receptor dimerization and is critical for mitogenic response stimulated by the growth factor (4, 6, 8–10).On the other hand, several authors working on FGF1 and FGF2 have suggested that there is no mandatory requirement for heparin for the assembly and activation of the FGF·FGFR complex. They imply that heparin only plays a role in association of two molecules of the growth factor and therefore facilitates their binding to FGFR (11). It has been reported that FGF1 and FGF2 can interact with the FGFR and trigger phosphorylation of p42/44 MAPK and activation of other signaling pathways even in the absence of HS (12–16).The accepted role of heparin/HS in FGF1 signaling is to prevent the degradation of the growth factor (17). The interaction with heparin or HS protects FGF1 against heat, acidic pH, and proteases (18, 19). HS also seems to regulate the activity of different FGFs by creating their local reservoir and generating a concentration gradient of the growth factor (6, 17).The binding of FGF1 to heparin/HS is mediated by specific residues forming a positively charged patch on the protein surface (20, 21). The major contribution is made by Lys¹¹⁸ (Lys¹³² in the full-length numbering system), which was identified by Harper and Lobb (22), and Lys¹¹² and Arg¹²² (23, 24). Additional residues of FGF1 involved in the interaction with heparin are the positively charged Lys¹¹³, Arg¹¹⁹, and Lys¹²⁸ and the polar Asn¹⁸, Asn¹¹⁴, and Gln¹²⁷ (20, 21). Site-directed mutagenesis and other studies have revealed the importance of Lys¹¹⁸ not only in heparin binding but also for the biological function of FGF1 (22, 25, 26). It was shown that the K118E (K132E) mutant is inactive in stimulation of DNA synthesis, although its affinity for FGFR and the ability to activate signaling cascades is not reduced (27, 28). Despite extensive research, the reason for the lack of mitogenic potential of K118E FGF1 is still not clear.In this paper, we verified the function of heparin in FGF1·FGFR complex formation and signaling by constructing several FGF1 mutants with reduced affinity for heparin. To recover the stability of these variants, which could no longer be stabilized by heparin, we supplemented them stepwise with stabilizing mutations (29). We analyzed thoroughly their biological activity and their ability to translocate across cellular membranes (30–34). Interestingly, the full mitogenic activity of the K118E FGF1 variant was restored by the introduced stabilizing mutations.Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat denaturation and proteolytic degradation and that the increased stability of the growth factor can compensate for reduced heparin binding.     

Synthesis of Free and Proliferating Cell Nuclear Antigen-bound Polyubiquitin Chains by the RING E3 Ubiquitin Ligase Rad5

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys⁶³-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13∼Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys⁶³ of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.DNA is susceptible to chemical alteration by many endogenous and exogenous agents. To counter this threat and maintain genome integrity, eukaryotic cells employ three main strategies: DNA repair pathways that directly reverse DNA damage, cell cycle checkpoints that allow time to repair the damage prior to replication, and DNA damage tolerance (DDT),² which is a method of bypassing DNA damage lesions during the DNA replication phase of the cell cycle.Proliferating cell nuclear antigen (PCNA) is a key regulatory protein in DNA replication and repair (1). At the replication fork, DNA is encircled by PCNA, a homotrimeric protein that promotes processive movement of the replicative DNA polymerase. Upon DNA damage and subsequent stalling of the replicative polymerase, Ub modifications of PCNA signal DDT, which allows a cell to bypass the lesion and proceed past this potential block in replication (2–4).In the DDT pathway, as in other Ub-dependent pathways, Ub is conjugated to a substrate by the actions of three enzymes, an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (5). The E1 enzyme initiates the pathway in a two-step reaction that utilizes ATP hydrolysis to activate the C terminus of Ub, culminating in the formation of an E1∼Ub thiolester. Subsequent transthiolation to the active site cysteine of the E2 generates an E2∼Ub thiolester. An E3 ligase then brings a substrate into close proximity to the E2∼Ub intermediate, thereby catalyzing the formation of an isopeptide bond between the amino group of a substrate lysine and the C-terminal glycine of Ub. Polyubiquitination occurs when this substrate is another Ub, either free or as part of a Ub-protein conjugate.The DDT pathway is characterized by distinct ubiquitination events on PCNA that occur in two stages (3, 4, 6). The first of these is monoubiquitination of lysine 164 on one or more of the PCNA subunits by the E2-E3 complex of Rad6-Rad18 in Saccharomyces cerevisiae (3, 4, 7). monoUb-PCNA can serve either as a signal for error-prone bypass of the DNA lesion by recruiting translesion polymerases or as a substrate for subsequent polyubiquitination by the E2 heterodimer Ubc13-Mms2 and the E3 ligase Rad5 (3, 4, 8, 9). The polyUb chain extended from the initial Ub moiety on monoUb-PCNA is linked specifically through Ub Lys⁶³ residues. This Lys⁶³-linked chain is thought to enable a template switch mechanism that allows for error-free bypass of the DNA lesion, in part by utilizing the single-strand DNA-dependent helicase activity of Rad5 (3, 4, 10, 11). Both PCNA ubiquitination events promote bypass of the DNA lesion rather than direct removal or repair of the lesion.We have been interested in the mechanism by which the yeast (Ubc13-Mms2)-Rad5 complex catalyzes the formation of Lys⁶³-linked polyUb on PCNA. Previous studies have shown that heterodimerization of the Ubc13-Mms2 E2 is essential for Lys⁶³-specific Ub-Ub conjugation in vitro and in vivo (12–15). Ubc13 is a canonical E2 enzyme with an active site cysteine that receives activated Ub by transthiolation from the E1∼Ub complex (12, 13). This Ub is referred to as the “donor Ub.” Mms2 is a Ub E2 variant protein that lacks the active site cysteine (12, 15); rather, Mms2 binds to a second Ub, the “acceptor Ub,” and positions it to facilitate nucleophilic attack on the Ubc13∼Ub thiolester bond by the ϵ-amine of Lys⁶³ (15, 16). The positioning of the acceptor Ub by Mms2 controls the specificity of polyUb assembly such that only Lys⁶³-linked chains can be formed (16).Ubc13-Mms2 can synthesize Lys⁶³-linked chains in vitro in the absence of a PCNA substrate or an E3 ligase (12, 13). However, unlike the synthesis of free Lys⁶³-linked polyUb chains by Ubc13-Mms2, little is known about the polyubiquitination of PCNA or the role of the Rad5 E3 ligase in these reactions. Rad5 can bind PCNA and Rad18, and it contains a catalytic RING domain that characterizes the largest class of E3 ligases (17–21). There is evidence that RING E3s like Rad5 may play a more active role in ubiquitination than simply bringing the substrate into close proximity with the E2∼Ub. Several RING E3s have been shown to stimulate the synthesis of unanchored polyUb chains or autoubiquitination of their cognate E2s in the absence of substrates (22–24). This stimulation may be related to the ability of RING E3s to enhance reactivity of the E2∼Ub thiolester bond through allosteric effects (25, 26).Using purified recombinant forms of Ubc13, Mms2, and Rad5, we have explored the assembly of free Lys⁶³-linked polyUb chains as well as the extension of a polyUb chain on a synthetic analog of monoUb-PCNA. We show that Rad5 facilitates ubiquitination in part by increasing the reactivity of the Ubc13∼Ub thiolester bond. With monoUb-PCNA substrates, Rad5 also stimulated polyubiquitination through direct interactions with PCNA and recruitment of Ub-charged Ubc13-Mms2. Surprisingly, Rad5 recognition of monoUb-PCNA appeared to depend on interactions only with the PCNA moiety of the conjugate, which suggests that substrate selectivity in vivo is likely to depend on additional factors.     

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN^{K252 + 255A}, SEN^K304A, SEN^K334A, SEN^K344E, SEN^K435L, and SEN^Δ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN^K344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN^K344E had lost α-enolase activity, which was also reduced in SEN^K334A and SEN^Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN^{K252 + 255A}, SEN^K435L, and SEN^Δ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.Streptococcus pyogenes (group A Streptococcus, GAS)⁸ is a common bacterial pathogen, causing over 700 million human disease episodes each year (1). These range from serious life-threatening invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome to non-invasive infections like pharyngitis and pyoderma. Invasive disease, in combination with postinfection immune sequelae including rheumatic heart disease and acute poststreptococcal glomerulonephritis, account for over half a million deaths each year (1). Although a resurgence of GAS invasive infections has occurred in western countries since the mid-1980s, disease burden is much greater in developing countries and indigenous populations of developed nations, where GAS infections are endemic (2–4).GAS is able to bind human plasminogen and activate the captured zymogen to the serine protease plasmin (5–17). The capacity of GAS to do this plays a critical role in virulence and invasive disease initiation (3, 17–19). The plasminogen activation system in humans is an important and highly regulated process that is responsible for breakdown of extracellular matrix components, dissolution of blood clots, and cell migration (20, 21). Plasminogen is a 92-kDa zymogen that circulates in human plasma at a concentration of 2 μm (22). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg⁵⁶¹–Val⁵⁶² site (23), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (20, 23). GAS also has the ability to activate human plasminogen by secreting the virulence determinant streptokinase. Streptokinase forms stable complexes with plasminogen or plasmin, both of which exhibit plasmin activity (20, 24). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α₂-antiplasmin and α₂-macroglobulin (11, 20). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (6). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (25), the PAM-related protein (19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (9, 26), and streptococcal surface enolase (SEN or α-enolase) (27). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (6).In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (27–29). SEN is abundantly expressed in the cytosol of most bacterial species but has also been identified as a surface-located protein in GAS and other bacteria including pneumococci, despite lacking classical cell surface protein motifs such as a signal sequence, membrane-spanning domain, or cell-wall anchor motif (27, 28, 30, 31). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (27, 32). In contrast, an internal binding motif containing lysines at positions 252 and 255 in the closely related α-enolase of Streptococcus pneumoniae has been shown to play a pivotal role in the acquisition of plasminogen in this bacterial species (33). The octameric pneumococcal α-enolase structure consists of a tetramer of dimers. Hence, potential binding sites could be buried in the interface between subunits. In fact, the crystal structure of S. pneumoniae α-enolase revealed that the two C-terminal lysine residues are significantly less exposed than the internal plasminogen-binding motif (34).In this study, we constructed an in silico model of GAS SEN, based on the pneumococcal octameric α-enolase crystal structure, and validated this model using ion mobility (IM) mass spectrometry (MS). Site-directed mutagenesis followed by structural and functional analyses revealed that Lys³⁴⁴ plays a crucial role in structural integrity and enzymatic function. Furthermore, we demonstrate that the plasminogen-binding motif residues Lys²⁵² and Lys²⁵⁵ and the C-terminal Lys⁴³⁴ and Lys⁴³⁵ residues are located adjacently in the GAS SEN structure and play a concerted role in the binding of human plasminogen.     

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.     

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.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

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.     

Protein-tyrosine Phosphatase-�� and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling

Calcium (Ca²⁺) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca²⁺ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca²⁺ release channel IP₃R. In response to IL-1, catalytically active PTPα was required for Ca²⁺ release from the ER, Src-dependent phosphorylation of IP₃R1 and accumulation of IP₃R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP₃R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca²⁺ signaling.The interleukin-1 (IL-1)³ family of pro-inflammatory cytokines mediates host responses to infection and injury. Impaired control of IL-1 signaling leads to chronic inflammation and destruction of extracellular matrices (1, 2), as seen in pathological conditions such as pulmonary fibrosis (3), rheumatoid arthritis (4, 5), and periodontitis (6). IL-1 elicits multiple signaling programs, some of which trigger Ca²⁺ release from the endoplasmic reticulum (ER) as well as expression of multiple cytokines and inflammatory factors including c-Fos and c-Jun (7, 8), and matrix metalloproteinases (9, 10), which mediate extracellular matrix degradation via mitogen-activated protein kinase-regulated pathways (11).In anchorage-dependent cells including fibroblasts and chondrocytes, focal adhesions (FAs) are required for IL-1-induced Ca²⁺ release from the ER and activation of ERK (12–14). FAs are actin-enriched adhesive domains composed of numerous (>50) scaffolding and signaling proteins (15–17). Many FA proteins are tyrosine-phosphorylated, including paxillin, focal adhesion kinase, and src family kinases, all of which are crucial for the assembly and disassembly of FAs (18–21). Protein-tyrosine phosphorylation plays a central role in regulating many cellular processes including adhesion (22, 23), motility (24), survival (25), and signal transduction (26–29). Phosphorylation of proteins by kinases is balanced by protein-tyrosine phosphatases (PTP), which can enhance or attenuate downstream signaling by dephosphorylation of tyrosine residues (30–32).PTPs can be divided into two main categories: receptor-like and intracellular PTPs (33). Two receptor-like PTPs have been localized to FA (leukocyte common antigen-related molecule and PTPα). Leukocyte common antigen-related molecule can dephosphorylate and mediate degradation of p130^cas, which ultimately leads to cell death (34, 35). PTPα contains a heavily glycosylated extracellular domain, a transmembrane domain, and two intracellular phosphatase domains (33, 36). The amino-terminal domain predominantly mediates catalytic activity, whereas the carboxyl-terminal domain serves a regulatory function (37, 38). PTPα is enriched in FA (23) and is instrumental in regulating FA dynamics (39) via activation of c-Src/Fyn kinases by dephosphorylating the inhibitory carboxyl tyrosine residue, namely Tyr⁵²⁹ (22, 40–42) and facilitation of integrin-dependent assembly of Src-FAK and Fyn-FAK complexes that regulate cell motility (43). Although PTPα has been implicated in formation and remodeling of FAs (44, 45), the role of PTPα in FA-dependent signaling is not defined.Ca²⁺ release from the ER is a critical step in integrin-dependent IL-1 signal transduction and is required for downstream activation of ERK (13, 46). The release of Ca²⁺ from the ER depends on the inositol 1,4,5-triphosphate receptor (IP₃R), which is an IP₃-gated Ca²⁺ channel (47). All of the IP₃R subtypes (subtypes 1–3) have been localized to the ER, as well as other the plasma membrane and other endomembranes (48–50). Further, IP₃R may associate with FAs, enabling the anchorage of the ER to FAs (51, 52). However, the molecule(s) that provide the structural link for this association has not been defined.FA-restricted, IL-1-triggered signal transduction in anchorage-dependent cells may rely on interacting proteins that are enriched in FAs and the ER (53). Here, we examined the possibility that PTPα associates with c-Src and IP₃R to functionally link FAs to the ER, thereby enabling IL-1 signal transduction.     

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.     

Queuosine Formation in Eukaryotic tRNA Occurs via a Mitochondria-localized Heteromeric Transglycosylase

tRNA guanine transglycosylase (TGT) enzymes are responsible for the formation of queuosine in the anticodon loop (position 34) of tRNA^Asp, tRNA^Asn, tRNA^His, and tRNA^Tyr; an almost universal event in eubacterial and eukaryotic species. Despite extensive characterization of the eubacterial TGT the eukaryotic activity has remained undefined. Our search of mouse EST and cDNA data bases identified a homologue of the Escherichia coli TGT and three spliced variants of the queuine tRNA guanine transglycosylase domain containing 1 (QTRTD1) gene. QTRTD1 variant_1 (Qv1) was found to be the predominant adult form. Functional cooperativity of TGT and Qv1 was suggested by their coordinate mRNA expression in Northern blots and from their association in vivo by immunoprecipitation. Neither TGT nor Qv1 alone could complement a tgt mutation in E. coli. However, transglycosylase activity could be obtained when the proteins were combined in vitro. Confocal and immunoblot analysis suggest that TGT weakly interacts with the outer mitochondrial membrane possibly through association with Qv1, which was found to be stably associated with the organelle.Queuosine (Q³; (7-{[(4,5-cis-dihydroxy-2-cyclo-penten-1-yl)-amino]methyl}-7-deazaguanosine) is a modified 7-deazaguanosine molecule found at the wobble position of transfer RNA that contains a GUN anticodon sequence: tRNA^Tyr, tRNA^Asn, tRNA^His, and tRNA^Asp (1). The Q-modification is widely distributed in nature in the tRNA of eubacteria, plants, and animals; a notable exception being yeast and plant leaf cells (2, 3). Interestingly, Q-modification has also been detected in aspartyl tRNA from mitochondria of rat (4) and opossum (5). In most eukaryotes, the Q molecule can be further modified by the addition of a mannosyl group to Q-tRNA^Asp and a galactosyl group to Q-tRNA^Tyr (1).Eubacteria are unique in their ability to synthesize Q. As part of this biosynthetic process, the eubacterial tRNA guanine transglycosylase (TGT) enzyme inserts the Q precursor molecule, 7-aminomethyl-7-deazaguanine (preQ₁) into tRNA, which is then converted to Q by two further enzymatic steps at the tRNA level (6). Eukaryotes by contrast salvage queuosine from food and enteric bacteria either as the free base (referred to as queuine) or as queuosine 5′-phosphate subsequent to normal tRNA turnover (7). A Q-related molecule, archaeosine, is found at position 15 of the D loop of most archaeal tRNA, where it functions to stabilize the tRNA structure (8). The enzyme involved in archaeosine biosynthesis is structurally and mechanistically related to the eubacterial TGT but with adaptations necessitated by the differences imposed by its unique substrate and tRNA specificity (9, 10).The crystal structure of the Zymononas mobilis (Z. mobilis) TGT has been determined and revealed the enzyme to be an irregular (β/α)₈ TIM barrel with a C-terminal zinc-binding subdomain (11). Insight into the residues involved in catalysis came from mutational and kinetic analysis of the recombinant Escherichia coli enzyme (12) and from the Z. mobilis TGT structure as an RNA-bound intermediate complexed to the final preQ₁-modified RNA product (13). This work showed the essential role of Asp-280 (Z. mobilis numbering) as the active site nucleophile. Asp-102, which was originally ascribed the role of active site nucleophile, functions as a general acid/base during catalysis (12, 10). Although, the E. coli and Z. mobilis TGT enzymes are monomeric in solution (14), at high protein concentrations the E. coli enzyme can oligomerize (15), and structural data from the Z. mobilis TGT has shown the formation of a 2:1 complex with tRNA; a possible functional requirement for catalysis (10).In contrast to the eubacterial enzyme, which is a single protein species, purification of the eukaryotic TGT suggested that the catalytically active enzyme is a heterodimeric molecule: subunits of 60 and 43 kDa in rabbit erythrocytes (16), 66 and 32 kDa in bovine liver (17), 60 and 34.5 kDa in rat liver (18), and a homodimer of two 68-kDa proteins in wheat germ (16, 19). A partial amino acid sequence was recovered from two of these active enzyme preparations. The identity of the proteins from bovine liver (17) could not be assigned at the time of publication. However, our searches show that the peptides from the larger 65-kDa subunit are identical to asparaginyl tRNA synthetase, and those of the smaller 32-kDa subunit correspond to 2,4-dienoyl CoA reductase. A highly pure preparation from rabbit reticulocytes (20) gave peptides with homology to the immunophilin p59, human elongation factor 2 (EF2), and a deubiquitinating enzyme, USP14. It is noteworthy that none of the peptide sequences obtained showed similarity to the eubacterial TGT. The results do suggest, however, that in eukaryotes the TGT activity could be embedded in a multisubunit complex.Most recently, Deshpande and Katze (21) identified a cDNA clone encoding a putative TGT catalytic subunit. Cloning the cDNA into a mammalian expression plasmid reconstituted TGT activity in GC₃/c1 cells, which are known to be naturally deficient in Q-containing tRNA (22). In this study, we identify for the first time the composition of the eukaryotic tRNA guanine transglycosylase, reconstitute the catalytic activity in vitro, and examine the intracellular distribution of the active subunits.