Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Structural Characterization of Monomeric/Dimeric State of p59fyn SH2 Domain.

Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59fyn SH2 domain.

Dynamically Coupled Residues within the SH2 Domain of FYN Are Key to Unlocking Its Activity.

Src kinase activity is controlled by various mechanisms involving a coordinated movement of kinase and regulatory domains. Notwithstanding the extensive knowledge related to the backbone dynamics, little is known about the more subtle side-chain dynamics within the regulatory domains and their role in the activation process. Here, we show through experimental methyl dynamic results and predicted changes in side-chain conformational couplings that the SH2 structure of Fyn contains a dynamic network capable of propagating binding information. We reveal that binding the phosphorylated tail of Fyn perturbs a residue cluster near the linker connecting the SH2 and SH3 domains of Fyn, which is known to be relevant in the regulation of the activity of Fyn. Biochemical perturbation experiments validate that those residues are essential for inhibition of Fyn, leading to a gain of function upon mutation. These findings reveal how side-chain dynamics may facilitate the allosteric regulation of the different members of the Src kinase family.

Structural insights into the intertwined dimer of fyn SH2.

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide-bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2's dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand.

Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding.

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses. The mazEF module encodes the MazF toxin and its cognate MazE antitoxin. The highly dynamic MazE possesses an N-terminal DNA binding domain through which it can negatively regulate its own promoter. Despite being one of the first TA systems studied, transcriptional regulation of Escherichia coli mazEF remains poorly understood. This paper presents the solution structure of C-terminal truncated E. coli MazE and a MazE-DNA model with a DNA palindrome sequence ∼ 10 bp upstream of the mazEF promoter. The work has led to a transcription regulator-DNA model, which has remained elusive thus far in the E. coli toxin-antitoxin family. Multiple complementary techniques including NMR, SAXS and ITC show that the long intrinsically disordered C-termini in MazE, required for MazF neutralization, does not affect the interactions between the antitoxin and its operator. Rather, the MazE C-terminus plays an important role in the MazF binding, which was found to increase the MazE affinity for the palindromic single site operator.

Structural and biophysical characterization of Staphylococcus aureus SaMazF shows conservation of functional dynamics.

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE.

Small-angle X-ray scattering- and nuclear magnetic resonance-derived conformational ensemble of the highly flexible antitoxin PaaA2.

Antitoxins from prokaryotic type II toxin-antitoxin modules are characterized by a high degree of intrinsic disorder. The description of such highly flexible proteins is challenging because they cannot be represented by a single structure. Here, we present a combination of SAXS and NMR data to describe the conformational ensemble of the PaaA2 antitoxin from the human pathogen E. coli O157. The method encompasses the use of SAXS data to filter ensembles out of a pool of conformers generated by a custom NMR structure calculation protocol and the subsequent refinement by a block jackknife procedure. The final ensemble obtained through the method is validated by an established residual dipolar coupling analysis. We show that the conformational ensemble of PaaA2 is highly compact and that the protein exists in solution as two preformed helices, connected by a flexible linker, that probably act as molecular recognition elements for toxin inhibition.

Study of the structural and dynamic effects in the FimH adhesin upon α-d-heptyl mannose binding.

Uropathogenic Escherichia coli cause urinary tract infections by adhering to mannosylated receptors on the human urothelium via the carbohydrate-binding domain of the FimH adhesin (FimHL). Numerous α-d-mannopyranosides, including α-d-heptyl mannose (HM), inhibit this process by interacting with FimHL. To establish the molecular basis of the high-affinity HM binding, we solved the solution structure of the apo form and the crystal structure of the FimHL-HM complex. NMR relaxation analysis revealed that protein dynamics were not affected by the sugar binding, yet HM addition promoted protein dimerization, which was further confirmed by small-angle X-ray scattering. Finally, to address the role of Y48, part of the "tyrosine gate" believed to govern the affinity and specificity of mannoside binding, we characterized the FimHL Y48A mutant, whose conformational, dynamical, and HM binding properties were found to be very similar to those of the wild-type protein.

¹H, ¹³C and ¹5N backbone and side-chain chemical shift assignments of the free and bound forms of the human PTPN11 second SH2 domain.

Src homology 2 (SH2) domains have an important role in the regulation of protein activity and intracellular signaling processes. They are geared to bind to specific phosphotyrosine (pY) motifs, with a substrate sequence specificity depending on the three amino acids immediately C-terminal to the pY. Here we report for the first time the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments for the C-terminal SH2 domain of the human protein tyrosine phosphatase PTPN11, both in its free and bound forms, where the ligand in the latter corresponds to a specific sequence of the human erythropoietin receptor.

The structure of TAX1BP1 UBZ1+2 provides insight into target specificity and adaptability.

TAX1BP1 is a novel ubiquitin-binding adaptor protein involved in the negative regulation of the NF-kappaB transcription factor, which is a key player in inflammatory responses, immunity and tumorigenesis. TAX1BP1 recruits A20 to the ubiquitinated signaling proteins TRAF6 and RIP1, leading to their A20-mediated deubiquitination and the disruption of IL-1-induced and TNF-induced NF-kappaB signaling, respectively. The two zinc fingers localized at its C-terminus function as novel ubiquitin-binding domains (UBZ, ubiquitin-binding zinc finger). Here we present for the first time both the solution and crystal structures of two classical UBZ domains in tandem within the human TAX1BP1. The relative orientation of the two domains is slightly different in the X-ray structure with respect to the NMR structure, indicating some degree of conformational flexibility, which is rationalized by NMR relaxation data. The observed degree of flexibility and stability between the two UBZ domains might have consequences on the recognition mechanism of interacting partners.

Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli.

Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcb1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract.

Mycoredoxin-1 is one of the missing links in the oxidative stress defence mechanism of Mycobacteria.

To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) with a CGYC catalytic motif. With mycoredoxin-1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin-1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin-1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S-mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin-1, revealing a thioredoxin fold with a putative mycothiol-binding site. With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin-1 as a function of time and find that mycoredoxin-1 gets S-mycothiolated on its N-terminal nucleophilic cysteine. Mycoredoxin-1 has a redox potential of -218 mV and hydrogen bonding with neighbouring residues lowers the pKa of its N-terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin-1, better understanding of mycothiol-dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.

Alternative interactions define gyrase specificity in the CcdB family.

Toxin-antitoxin (TA) modules are small operons associated with stress response of bacteria. F-plasmid CcdB(F) was the first TA toxin for which its target, gyrase, was identified. Plasmidic and chromosomal CcdBs belong to distinct families. Conserved residues crucial for gyrase poisoning activity of plasmidic CcdBs are not conserved among these families. Here we show that the chromosomal CcdB(Vfi) from Vibrio fischeri is an active gyrase poison that interacts with its target via an alternative energetic mechanism. Changes in the GyrA14-binding surface of the Vibrio and F-plasmid CcdB family members illustrate neutral drift where alternative interactions can be used to achieve the same functionality. Differences in affinity between V. fischeri and F-plasmid CcdB for gyrase and their corresponding CcdA antitoxin possibly reflect distinct roles for TA modules located on plasmids and chromosomes.

Purification, crystallization and preliminary X-ray diffraction analysis of the Fyn SH2 domain and its complex with a phosphotyrosine peptide.

SH2 domains are widespread protein-binding modules that recognize phosphotyrosines and play central roles in intracellular signalling pathways. The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. X-ray data were collected to a resolution of 2.00 Å for the unbound form and 1.40 Å for the protein in complex with the phosphotyrosine peptide.

The plasticity of the ß-trefoil fold constitutes an evolutionary platform for protease inhibition.

Proteases carry out a number of crucial functions inside and outside the cell. To protect the cells against the potentially lethal activities of these enzymes, specific inhibitors are produced to tightly regulate the protease activity. Independent reports suggest that the Kunitz-soybean trypsin inhibitor (STI) family has the potential to inhibit proteases with different specificities. In this study, we use a combination of biophysical methods to define the structural basis of the interaction of papaya protease inhibitor (PPI) with serine proteases. We show that PPI is a multiple-headed inhibitor; a single PPI molecule can bind two trypsin units at the same time. Based on sequence and structural analysis, we hypothesize that the inherent plasticity of the ß-trefoil fold is paramount in the functional evolution of this family toward multiple protease inhibition.

Molecular identification of ß-citrylglutamate hydrolase as glutamate carboxypeptidase 3.

ß-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify ß-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed ß-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that ß-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of ß-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to ß-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed ß-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and ß-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze ß-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its ß-citrylglutamate hydrolase activity.

1H, 13C and 15N backbone and side-chain chemical shift assignment of the Fyn SH2 domain and its complex with a phosphotyrosine peptide.

SH2 domains are interaction modules uniquely dedicated to recognize phosphotyrosine sites, playing a central role in for instance the activation of tyrosine kinases or phosphatases. Here we report the (1)H, (15)N and (13)C backbone and side-chain chemical shift assignments of the SH2 domain of the human protein tyrosine kinase Fyn, both in its free state and bound to a high-affinity phosphotyrosine peptide corresponding to a specific sequence in the hamster middle-T antigen. The BMRB accession numbers are 17,368 and 17,369, respectively.

Hydrophobic surface patches on LolA of Pseudomonas aeruginosa are essential for lipoprotein binding.

Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA-lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open beta-barrel covered by alpha helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA-lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches.

Structural and thermodynamic characterization of Vibrio fischeri CcdB.

CcdB(Vfi) from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdB(Vfi) is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdB(Vfi) possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdB(Vfi) shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications.

Structural and thermodynamic characterization of pre- and postpolymerization states in the F4 fimbrial subunit FaeG.

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27 versus approximately 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.