Structural and computational investigations of VIM-7: insights into the substrate specificity of vim metallo-β-lactamases

The presence of metallo-β-lactamases (MBLs) in many clinically important human bacterial pathogens limits treatment options, as these enzymes efficiently hydrolyze nearly all β-lactam antibiotics. VIM enzymes are among the most widely distributed MBLs, but many of the individual VIM subtypes remain poorly characterized. Pseudomonas aeruginosa VIM-7 is the most divergent among VIM-type MBLs in terms of amino acid sequence. Here we present crystal structures of VIM-7 as the native enzyme, with Cys221 oxidized (VIM-7-Ox), and with a sulfur atom bridging the two active-site zinc ions (VIM-7-S). Comparison with VIM-2 and VIM-4 structures suggests an explanation for the reduced catalytic efficiency of VIM-7 against cephalosporins with a positively charged cyclic substituent at the C3 position (e.g., ceftazidime). Kinetic variations are attributed to substitutions in residues 60-66 (that form a loop adjacent to the active site previously implicated in substrate binding) and to the disruption of two hydrogen-bonding clusters through substitutions at positions 218 and 224. Furthermore, the less negatively charged surface of VIM-7 (compared to VIM-2) may also contribute to the reduced hydrolytic efficiency. Docking of the cephalosporins ceftazidime and cefotaxime into the VIM-2 and VIM-7 structures reveals that amino acid substitutions may cause the mode of substrate binding to differ between the two enzymes. Our structures thus provide new insights into the variation in substrate specificity that is evident across this family of clinically important enzymes.     

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity.We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl₂ or MnCl₂. In the E122Q mutant, k_cat is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.     

The capsid of the small RNA phage PRR1 is stabilized by metal ions

Many nonenveloped virus particles are stabilized by calcium ions bound in the interfaces between the protein subunits. These ions may have a role in the disassembly process. The small RNA phages of the Leviviridae family have T = 3 quasi-symmetry and are unique among simple viruses in that they have a coat protein with a translational repressor activity and a fold that has not been observed in other viruses. The crystal structure of phage PRR1 has been determined to 3.5 Å resolution. The structure shows a tentative binding site for a calcium ion close to the quasi-3-fold axis. The RNA-binding surface used for repressor activity is mostly conserved. The structure does not show any significant differences between quasi-equivalent subunits, which suggests that the assembly is not controlled by conformational switches as in many other simple viruses.     

Immunoglobulin G1 Fc Domain Motions: Implications for Fc Engineering

The fragment crystallizable (Fc) region links the key pathogen identification and destruction properties of immunoglobulin G (IgG). Pathogen opsonization positions Fcs to activate pro-inflammatory Fcγ receptors (FcγRs) on immune cells. The cellular response and committal to a damaging, though protective, immune response are tightly controlled at multiple levels. Control mechanisms are diverse and in many cases unclear, but one frequently suggested contribution originates in FcγR affinity being modulated through shifts in Fc conformational sampling. Here, we report a previously unseen IgG1 Fc conformation. This observation motivated an extensive molecular dynamics investigation of polypeptide and glycan motions that revealed greater amplitude of motion for the N-terminal Cγ2 domains and N-glycan than previously observed. Residues in the Cγ2/Cγ3 interface and disulfide-bonded hinge were identified as influencing the Cγ2 motion. Our results are consistent with a model of Fc that is structurally dynamic. Conformational states that are competent to bind immune-stimulating FcγRs interconverted with Fc conformations distinct from those observed in FcγR complexes, which may represent a transient, nonbinding population.     

Structure of an archaeal homolog of the human protein complex Rpp21-Rpp29 that is a key core component for the assembly of active ribonuclease P

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5′-leader sequence of precursor tRNA. Human RNase P protein subunits Rpp21 and Rpp29, which bind to each other, with catalytic RNA (H1 RNA) are sufficient for activating endonucleolytic cleavage of precursor tRNA. Here we have determined the crystal structure of the complex between the Pyrococcus horikoshii RNase P proteins PhoRpp21 and PhoRpp29, the archaeal homologs of Rpp21 and Rpp29, respectively. PhoRpp21 and PhoRpp29 form a heterodimeric structure where the two N-terminal helices (α1 and α2) in PhoRpp21 predominantly interact with the N-terminal extended structure, the β-strand (β2), and the C-terminal helix (α3) in PhoRpp29. The interface is dominated by hydrogen bonds and several salt bridges, rather than hydrophobic interactions. The electrostatic potential on the surface of the heterodimer shows a positively charged cluster on one face, suggesting a possible RNA-binding surface of the PhoRpp21-PhoRpp29 complex. The present structure, along with the result of a mutational analysis, suggests that heterodimerization between PhoRpp21 and PhoRpp29 plays an important role in the function of P. horikoshii RNase P.     

An allosteric circuit in caspase-1

Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface ∼ 15 Å from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them—Arg286 and Glu390, which form a salt bridge—have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k_cat/K_m). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k_cat/K_m. X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or “hot wire” that promotes site-to-site coupling.     

High-resolution crystal structures of trimeric and rod phycocyanin

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (L_R^8.7), the rod linker (L_R), and only one of three rod-core linkers (L_RC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the L_R protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.     

Statics of the Ribosomal Exit Tunnel: Implications for Cotranslational Peptide Folding, Elongation Regulation, and Antibiotics Binding

A sophisticated interplay between the static properties of the ribosomal exit tunnel and its functional role in cotranslational processes is revealed by constraint counting on topological network representations of large ribosomal subunits from four different organisms. As for the global flexibility characteristics of the subunit, the results demonstrate a conserved stable structural environment of the tunnel. The findings render unlikely that deformations of the tunnel move peptides down the tunnel in an active manner. Furthermore, the stable environment rules out that the tunnel can adapt widely so as to allow tertiary folding of nascent chains. Nevertheless, there are local zones of flexible nucleotides within the tunnel, between the peptidyl transferase center and the tunnel constriction, and at the tunnel exit. These flexible zones strikingly agree with previously identified folding zones. As for cotranslational elongation regulation, flexible residues in the β-hairpin of the ribosomal L22 protein were verified, as suggested previously based on structural results. These results support the hypothesis that L22 can undergo conformational changes that regulate the tunnel voyage of nascent polypeptides. Furthermore, rRNA elements, for which conformational changes have been observed upon interaction of the tunnel wall with a nascent SecM peptide, are less strongly coupled to the subunit core. Sequences of coupled rigid clusters are identified between the tunnel and some of these elements, suggesting signal transmission by a domino-like mechanical coupling. Finally, differences in the flexibility of the glycosidic bonds of bases that form antibiotics-binding crevices within the peptidyl transferase center and the tunnel region are revealed for ribosomal structures from different kingdoms. In order to explain antibiotics selectivity, action, and resistance, according to these results, differences in the degrees of freedom of the binding regions may need to be considered.     

Structural Basis of Conserved Cysteine in the Fibroblast Growth Factor Family: Evidence for a Vestigial Half-Cystine

The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in ∼ 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.     

Crystal Structure of Prostate Secretory Protein PSP94 Shows an Edge-to-Edge Association of two Monomers to Form a Homodimer

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 Å resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last β-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.     

Symmetry and asymmetry of the RING-RING dimer of Rad18

The human ubiquitin-conjugating enzyme Rad6 (E2), with ubiquitin ligase enzyme Rad18 (RING E3), monoubiquitinates proliferating cell nuclear antigen at stalled replication forks in DNA translesion synthesis. Here, we determine the structure of the homodimeric Rad18 RING domains by X-ray crystallography and classify it to RING-RING dimers that dimerize through helices adjacent to the RING domains and through the canonical RING domains. Using NMR spectroscopy and site-directed mutagenesis, we demonstrate that the Rad6b binding site, for the Rad18 RING domain, strongly resembles that of other E2/E3 RING/U-box complexes. We show that the homodimeric Rad18 RING domain can recruit two Rad6b E2 enzymes, whereas the full-length Rad18 homodimer binds only to a single Rad6b molecule. Such asymmetry is a common feature of RING-RING heterodimers and has been observed for the CHIP U-box homodimer. We propose that asymmetry may be a common feature of dimeric RING E3 ligases.     

Structures of Intermediates along the Catalytic Cycle of Terminal Deoxynucleotidyltransferase: Dynamical Aspects of the Two-Metal Ion Mechanism

Terminal deoxynucleotidyltransferase (Tdt) is a non-templated eukaryotic DNA polymerase of the polX family that is responsible for the random addition of nucleotides at the V(D)J junctions of immunoglobulins and T-cell receptors. Here we describe a series of high-resolution X-ray structures that mimic the pre-catalytic state, the post-catalytic state and a competent state that can be transformed into the two other ones in crystallo via the addition of dAMPcPP and Zn^2 +, respectively. We examined the effect of Mn^2 +, Co^2 + and Zn^2 + because they all have a marked influence on the kinetics of the reaction. We demonstrate a dynamic role of divalent transition metal ions bound to site A: (i) Zn^2 + (or Co^2 +) in Metal A site changes coordination from octahedral to tetrahedral after the chemical step, which explains the known higher affinity of Tdt for the primer strand when these ions are present, and (ii) metal A has to leave to allow the translocation of the primer strand and to clear the active site, a typical feature for a ratchet-like mechanism. Except for Zn^2 +, the sugar puckering of the primer strand 3′ terminus changes from C2′-endo to C3′-endo during catalysis. In addition, our data are compatible with a scheme where metal A is the last component that binds to the active site to complete its productive assembly, as already inferred in human pol beta. The new structures have potential implications for modeling pol mu, a closely related polX implicated in the repair of DNA double-strand breaks, in a complex with a DNA synapsis.     

Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-guanine transglycosylase: insight into the regulation of substrate selectivity by kinetic and crystallographic studies

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of guanine in the wobble position of particular tRNAs by the modified base preQ₁. In vitro, however, the enzyme is also able to insert the immediate biosynthetic precursor, preQ₀, into those tRNAs. This substrate promiscuity is based on a peptide switch in the active site, gated by the general acid/base Glu235. The switch alters the properties of the binding pocket to allow either the accommodation of guanine or preQ₁. The peptide conformer recognising guanine, however, is also able to bind preQ₀. To investigate selectivity regulation, kinetic data for Zymomonas mobilis Tgt were recorded. They show that selectivity in favour of the actual substrate preQ₁ over preQ₀ is not achieved by a difference in affinity but via a higher turnover rate. Moreover, a Tgt(Glu235Gln) variant was constructed. The mutation was intended to stabilise the peptide switch in the conformation favouring guanine and preQ₀ binding. Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln exchange has, with respect to all substrate bases, no significant influence on k_cat. In contrast, K_M(preQ₁) is drastically increased, while K_M(preQ₀) seems to be decreased. Hence, regarding k_cat/K_M as an indicator for catalytic efficiency, selectivity of Tgt in favour of preQ₁ is abolished or even inverted in favour of preQ₀ for Tgt(Glu235Gln). Crystal structures of the mutated enzyme confirm that the mutation strongly favours the binding pocket conformation required for the accommodation of guanine and preQ₀. The way this is achieved, however, significantly differs from that predicted based on crystal structures of wild-type Tgt.     

Identification and Characterization of a Misfolded Monomeric Serpin Formed at Physiological Temperature

The native serpin state is kinetically trapped. However, under mildly destabilizing conditions, the conformational landscape changes, and a number of nonnative conformations with increased stability can be readily formed. The ability to undergo structural change is due to intrinsic strain within the serpin's tertiary fold, which is utilized for proteinase inhibition but renders the protein susceptible to aberrant folding and self-association. The relationship between these various conformations is poorly understood. Antichymotrypsin (ACT) is an inhibitory serpin that readily forms a number of inactive conformations, induced via either environmental stress or interaction with proteinases. Here we have used a variety of biophysical and structural techniques to characterize the relationship between some of these conformations. Incubation of ACT at physiological temperature results in the formation of a range of conformations, including both polymer and misfolded monomer. The ability to populate these nonnative states and the native conformation reflects an energy landscape that is very sensitive to the solution conditions. X-ray crystallography reveals that the misfolded monomeric conformation is in the delta conformation. Further polymerization and seeding experiments show that the delta conformation is an end point in the misfolding pathway of ACT and not an on-pathway intermediate formed during polymerization. The observation that ACT readily forms this inactive conformation at physiological temperature and pH suggests that it may have a role in both health and disease.     

Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.     

The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C₄mim]Cl⁻. In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.     

Visualizing active-site dynamics in single crystals of HePTP: opening of the WPD loop involves coordinated movement of the E loop

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loop in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an ‘atypically open’ conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.