Structural differences between the Streptococcus agalactiae housekeeping and pilus-specific sortases: SrtA and SrtC1

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a 'lid' in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the 'lid' mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis.     

Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr¹⁶⁰ in SrtB to an arginine as in SrtC (Arg¹⁶⁰) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.     

Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity

Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two α-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.     

Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD⁺. A new phosphate recognition site, “new P_i” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical P_i” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “P_s” site in the ternary complexes but at the “new P_i” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new P_i” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism.     

First structures of an active bacterial tyrosinase reveal copper plasticity

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.     

Analysis of binding site hot spots on the surface of Ras GTPase

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.     

The Effects of Phosphomimetic Lid Mutation on the Thermostability of the N-terminal Domain of MDM2

The multidomain E3 ubiquitin ligase MDM2 catalyzes p53 ubiquitination by a “dual-site” docking mechanism whereby MDM2 binding to at least two distinct peptide motifs on p53 promotes ubiquitination. One protein-protein interaction occurs between the N-terminal hydrophobic pocket of MDM2 and the transactivation motif of p53, and the second interaction occurs between the acidic domain of MDM2 and a motif in the DNA-binding domain of p53. A flexible N-terminal pseudo-substrate or “lid” adjacent to the N-terminal hydrophobic pocket of MDM2 has a phosphorylation site, and there are distinct models proposed on how the phosphorylated lid could affect MDM2 function. Biochemical studies have predicted that phosphomimetic mutation will stabilize the lid on the surface of MDM2 and will “open” the hydrophobic pocket and stabilize the MDM2-p53 complex, while NMR studies proposed that phosphomimetic mutation “closes” the lid over the MDM2 pocket and inhibits MDM2-p53 complex formation. To resolve these discrepancies, we utilized a quantitative fluorescence-based dye binding assay to measure the thermal unfolding of wild-type (wt), ΔLid, and S17D N-terminal domains of MDM2 as a function of increasing ligand concentration. Our data reveal that S17D lid mutation increases, rather than decreases, the thermostability of the N-terminal domain of MDM2 in the absence or in the presence of ligand. ΔLid mutation, by contrast, increases MDM2 thermoinstability. This is consistent with biochemical data, using full-length MDM2, showing that the S17D mutation stabilizes the MDM2-p53 complex and increases the specific activity of the E3 ubiquitin ligase function of MDM2. These data indicate that phosphomimetic lid mutation results in an “opening,” rather than a “closing,” of the pocket of MDM2 and highlight the ability of small intrinsically disordered or unstructured peptide motifs to regulate the specific activity of a protein.     

Two Crystal Structures of Escherichia coli N-Acetyl-l-Glutamate Kinase Demonstrate the Cycling between Open and Closed Conformations

N-Acetyl-l-glutamate kinase (NAGK), the paradigm enzyme of the amino acid kinase family, catalyzes the second step of arginine biosynthesis. Although substrate binding and catalysis were clarified by the determination of four crystal structures of the homodimeric Escherichia coli enzyme (EcNAGK), we now determine 2 Å resolution crystal structures of EcNAGK free from substrates or complexed with the product N-acetyl-l-glutamyl-5-phosphate (NAGP) and with sulfate, which reveal a novel, very open NAGK conformation to which substrates would associate and from which products would dissociate. In this conformation, the C-domain, which hosts most of the nucleotide site, rotates ∼ 24°-28° away from the N-domain, which hosts the acetylglutamate site, whereas the empty ATP site also exhibits some changes. One sulfate is found binding in the region where the β-phosphate of ATP normally binds, suggesting that ATP is first anchored to the β-phosphate site, before perfect binding by induced fit, triggering the shift to the closed conformation. In contrast, the acetylglutamate site is always well formed, although its β-hairpin lid is found here to be mobile, being closed only in the subunit of the EcNAGK-NAGP complex that binds NAGP most strongly. Lid closure appears to increase the affinity for acetylglutamate/NAGP and to stabilize the closed enzyme conformation via lid-C-domain contacts. Our finding of NAGP bound to the open conformation confirms that this product dissociates from the open enzyme form and allows reconstruction of the active center in the ternary complex with both products, delineating the final steps of the reaction, which is shown here by site-directed mutagenesis to involve centrally the invariant residue Gly11.     

X-ray structures of Bacillus pallidusd-arabinose isomerase and its complex with l-fucitol

d-Arabinose isomerase (d-AI), also known as l-fucose isomerase (l-FI), catalyzes the aldose–ketose isomerization of d-arabinose to d-ribulose, and l-fucose to l-fuculose. Bacillus pallidus (B. pallidus) d-AI can catalyze isomerization of d-altrose to d-psicose, as well as d-arabinose and l-fucose. Three X-ray structures of B. pallidusd-AI in complexes with 2-methyl-2,4-pentadiol, glycerol and an inhibitor, l-fucitol, were determined at resolutions of 1.77, 1.60 and 2.60 Å, respectively. B. pallidusd-AI forms a homo-hexamer, and one subunit has three domains of almost equal size; two Rossmann fold domains and a mimic of the (β/α) barrel fold domain. A catalytic metal ion (Mn²⁺) was found in the active site coordinated by Glu342, Asp366 and His532, and an additional metal ion was found at the channel for the passage of a substrate coordinated by Asp453. The X-ray structures basically supported the ene-diol mechanism for the aldose–ketose isomerization by B. pallidusd-AI, as well as Escherichia coli (E. coli) l-FI, in which Glu342 and Asp366 facing each other at the catalytic metal ion transfer a proton from C2 to C1 and O1 to O2, acting as acid/base catalysts, respectively. However, considering the ionized state of Asp366, the catalytic reaction also possibly occurs through the negatively charged ene-diolate intermediate stabilized by the catalytic metal ion. A structural comparison with E. colil-FI showed that B. pallidusd-AI possibly interconverts between “open” and “closed” forms, and that the additional metal ion found in B. pallidusd-AI may help to stabilize the channel region.     

X-ray Crystallographic and MD Simulation Studies on the Mechanism of Interfacial Activation of a Family I.3 Lipase with Two Lids

The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two α-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 Å resolution in the presence of Ca²⁺ and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca²⁺-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca²⁺ and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.     

Substrate Binding and Catalysis in Carbamate Kinase Ascertained by Crystallographic and Site-Directed Mutagenesis Studies: Movements and Significance of a Unique Globular Subdomain of This Key Enzyme for Fermentative ATP Production in Bacteria

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 Å resolution and containing bound MgADP, and the other at 2.1 Å resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K_m^CP and k_cat triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.     

Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.     

Crystal structures of cobalamin-independent methionine synthase (MetE) from Streptococcus mutans: a dynamic zinc-inversion model

Cobalamin-independent methionine synthase (MetE) catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to l-homocysteine to form methionine. Previous studies have shown that the MetE active site coordinates a zinc atom, which is thought to act as a Lewis acid and plays a role in the activation of thiol. Extended X-ray absorption fine structure studies and mutagenesis experiments identified the zinc-binding site in MetE from Escherichia coli. Further structural investigations of MetE from Thermotoga maritima lead to the proposition of two models: “induced fit” and “dynamic equilibrium”, to account for the catalytic mechanisms of MetE. Here, we present crystal structures of oxidized and zinc-replete MetE from Streptococcus mutans at the physiological pH. The structures reveal that zinc is mobile in the active center and has the possibility to invert even in the absence of homocysteine. These structures provide evidence for the dynamic equilibrium model.     

The structure of LL-diaminopimelate aminotransferase from Chlamydia trachomatis: implications for its broad substrate specificity

We have previously reported the structures of the native holo and substrate-bound forms of ll-diaminopimelate aminotransferase from Arabidopsis thaliana (AtDAP-AT). Here, we report the crystal and molecular structures of the ll-diaminopimelate aminotransferase from Chlamydia trachomatis (CtDAP-AT) in the apo-form and the pyridoxal-5′-phosphate-bound form. The molecular structure of CtDAP-AT shows that its overall fold is essentially identical with that of AtDAP-AT except that CtDAP-AT adopts an “open” conformation as opposed to the “closed” conformation of AtDAP-AT. Although AtDAP-AT and CtDAP-AT are approximately 40% identical in their primary sequence, they have major differences in their substrate specificities; AtDAP-AT is highly specific for LL-DAP, whereas CtDAP-AT accepts a wider range of substrates. Since all of the residues involved in substrate recognition are highly conserved between AtDAP-AT and CtDAP-AT, we propose that differences in flexibility of the loops lining the active-site region between the two enzymes likely account for the differences in substrate specificity.     

Phage display and crystallographic analysis reveals potential substrate/binding site interactions in the protein secretion chaperone CsaA from Agrobacterium tumefaciens

The protein CsaA has been proposed to function as a protein secretion chaperone in bacteria that lack the Sec-dependent protein-targeting chaperone SecB. CsaA is a homodimer with two putative substrate-binding pockets, one in each monomer. To test the hypothesis that these cavities are indeed substrate-binding sites able to interact with other polypeptide chains, we selected a peptide that bound to CsaA from a random peptide library displayed on phage. Presented here is the structure of CsaA from Agrobacterium tumefaciens (AtCsaA) solved in the presence and absence of the selected peptide. To promote co-crystallization, the sequence for this peptide was genetically fused to the amino-terminus of AtCsaA. The resulting 1.65 Å resolution crystal structure reveals that the tethered peptide from one AtCsaA molecule binds to the proposed substrate-binding pocket of a symmetry-related molecule possibly mimicking the interaction between a pre-protein substrate and CsaA. The structure shows that the peptide lies in an extended conformation with alanine, proline and glutamine side chains pointing into the binding pocket. The peptide interacts with the atoms of the AtCsaA-binding pocket via seven direct hydrogen bonds. The side chain of a conserved pocket residue, Arg76, has an “up” conformation when the CsaA-binding site is empty and a “down” conformation when the CsaA-binding site is occupied, suggesting that this residue may function to stabilize the peptide in the binding cavity. The presented aggregation assays, phage-display analysis and structural analysis are consistent with AtCsaA being a general chaperone. The properties of the proposed CsaA-binding pocket/peptide interactions are compared to those from other structurally characterized molecular chaperones.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

The structural basis for the activation and peptide recognition of bacterial ClpP

ClpP and its ATPase compartment, ClpX or ClpA, remove misfolded proteins in cells and are of utmost importance in protein quality control. The ring hexamers of ClpA or ClpX recognize, unfold, and translocate target substrates into the degradation chamber of the double-ring tetradecamer of ClpP. The overall reaction scheme catalyzed by ClpXP or ClpAP has been proposed; however, the molecular mechanisms associated with substrate recognition and degradation have not yet been clarified in detail. To investigate these mechanisms, we determined the crystal structures of ClpP from Helicobacter pylori in complex with product peptides bound to the active site as well as in the apo state. In the complex structure, the peptides are zipped with two antiparallel strands of ClpP and point to the adjacent active site, thus providing structural explanations for the broad substrate specificity, the product inhibition and the processive degradation of substrates in the chamber. The structures also suggest that substrate binding causes local conformational changes around the active site that ultimately induce the active conformation of ClpP.     

The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX₃C” or twin “CX₉C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX₂C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.     

Crystal Structure of the Mycoplasma arthritidis-Derived Mitogen in Apo Form Reveals a 3D Domain-Swapped Dimer

Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing particular Vβ elements of T cell receptor. Here, we report the crystal structure of a MAM mutant K201A in apo form (unliganded) at 2.8-Å resolutions. We also partially refined the crystal structures of the MAM wild type and another MAM mutant L50A in apo forms at low resolutions. Unexpectedly, the structures of these apo MAM molecules display a three-dimensional domain-swapped dimer. The entire C-terminal domains of these MAM molecules are involved in the domain swapping. Functional analyses demonstrated that the K201A and L50A mutants do not show altered ability to bind to their host receptors and that they stimulate the activation of T cells as efficiently as does the wild type. Structural comparisons indicated that the “reconstituted” MAM monomer from the domain-swapped dimer displays large differences at the hinge regions from the MAM_wt molecule in the receptor-bound form. Further comparison indicated that MAM has a flexible N-terminal loop, implying that conformational changes could occur upon receptor binding.     

Evolution of Stability in a Cold-Active Enzyme Elicits Specificity Relaxation and Highlights Substrate-Related Effects on Temperature Adaptation

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 °C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity.