[Show abstract][Hide abstract] ABSTRACT: Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signalling 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 behaviour, on the other hand. This article is protected by copyright. All rights reserved.
Protein Science 09/2015; DOI:10.1002/pro.2806 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HigA2 antitoxin and the HigBA2 toxin-antitoxin complex from Vibrio cholerae were crystallized in complex with their operator box. Screening of 22 different DNA duplexes led to two crystal forms of HigA2 complexes and one crystal form of a HigBA2 complex. Crystals of HigA2 in complex with a 17 bp DNA duplex belong to space group P3221, with unit-cell parameters a = b = 94.0, c = 123.7 Å, and diffract to 2.3 Å resolution. The second form corresponding to HigA2 in complex with a 19 bp duplex belong to space group P43212 and only diffract to 3.45 Å resolution. Crystals of the HigBA2 toxin-antitoxin were obtained in complex with a 31 bp duplex and belonged to space group P41212, with unit-cell parameters a = b = 113.6, c = 121.1 Å. They diffract to 3.3 Å resolution.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2015; 71(Pt 2):226-33. DOI:10.1107/S2053230X15000746 · 0.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription
of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation
of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53
binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous
p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second
but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds
opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the
p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and
chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target
genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional
modifications account for these observations.
Nucleic Acids Research 10/2014; 42(20). DOI:10.1093/nar/gku962 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we
show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd,
Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop
acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized
[Show abstract][Hide abstract] ABSTRACT: Originally identified as plasmid-stabilizing entities, toxin-antitoxin (TA) modules are ubiquitous in the genomes of prokaryotes and archeae. Most commonly they constitute small operons that encode two genes. The downstream gene encodes for a toxic protein, while the upstream 'antitoxin' gene protects the cell against this toxin. TA antitoxins typically contain a significant amount of intrinsic disorder, with variable degrees of prestructuring. The main function of the IDP segments is folding-upon-binding, and this functionality is directly linked to the evolutionary origin of TA modules. Therefore, in order to understand the nature and functionality of intrinsic disorder in antitoxins, one first has to look at the architectures of type II TA modules and how they likely evolved. One may consider a regulatory segment breaking off from a toxin precursor and fusing to a common transcription regulator domain, thus creating a module with novel regulatory properties.
Chemical Reviews 05/2014; 114(13). DOI:10.1021/cr400656f · 46.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 04/2014; 42(10). DOI:10.1093/nar/gku266 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein
translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble
uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis
is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure
of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the
tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these
conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free
state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization
of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and
coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization
of MnmEG form an integral part of the tRNA modification reaction cycle.
Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku213 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fic enzymes post-translationally modify proteins through AMPylation, UMPylation, phosphorylation, or phosphocholination. They have been identified across all domains of life, and they target a myriad of proteins such as eukaryotic GTPases, unstructured protein segments, and bacterial enzymes. Consequently, they play crucial roles in eukaryotic signal transduction, drug tolerance, bacterial pathogenicity, and the bacterial stress response. Structurally, they consist of an all α-helical core domain that supports and scaffolds a structurally conserved active-site loop, which catalyses the transfer of various parts of a nucleotide cofactor to proteins. Despite their diverse substrates and targets, they retain a conserved active site and reaction chemistry. This catalytic variety came to light only recently with the crystal structures of different Fic enzymes.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 signalling proteins TRAF6 and RIP1, leading to their A20-mediated deubiquitination and the disruption of IL-1 and TNF-induced NF-kappaB signalling, respectively. The two zinc fingers localized at its C-terminal, 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.
[Show abstract][Hide abstract] ABSTRACT: The phd/doc family is one the smallest families of toxin–antitoxin modules and was first discovered as a plasmid addiction module on E. coli bacteriophage P1. The toxin Doc interacts with the ribosome, competes with hygromycin, and inhibits translation. Structurally, Doc resembles Fic domains, which are known to transfer an AMP moiety to the hydroxyphenyl group of a tyrosine in the target protein. Although much of the AMP/ATP binding site of Fic is conserved in Doc, no specific enzymatic activity has yet been linked to Doc. Nevertheless, mutations in the loop corresponding to the active site loop of Fic render Doc inactive. Regulation of the P1 phd/doc operon is understood in terms of a detailed molecular mechanism and involves conditional cooperativity. The N-terminal domain of Phd forms the DNA-binding unit and represents a common DNA-binding fold that is also shared with a number of antitoxins from different TA families, among which YefM is the best studied. Enhancement of the DNA-binding affinity of Phd by Doc stems both from allosteric coupling between the Doc- and DNA-binding sites on Phd and from avidity effects due to Doc-mediated bridging of two Phd dimers bound to the operator site. Activation of the system at high Doc-to-Phd ratios stems from a low-to-high affinity switch in the interaction between Phd and Doc.
[Show abstract][Hide abstract] ABSTRACT: Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of the catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.
Nature Chemical Biology 10/2013; 9(12). DOI:10.1038/nchembio.1364 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex crystallized in two crystal forms. Crystals of form I belonged to space group P21212, with unit-cell parameters a = 129.0, b = 119.8, c = 33.4 Å, and diffracted to 3.0 Å resolution. The asymmetric unit is likely to contain a single complex consisting of two toxin monomers and one antitoxin dimer. The second crystal form crystallized in space group P3221, with unit-cell parameters a = 134.5, c = 55.4 Å. These crystals diffracted to 2.2 Å resolution and probably contain a complex with a different stoichiometry. Crystals of the C-terminal domain of HigA2 belonged to space group C2, with unit-cell parameters a = 115.4, b = 61.2, c = 73.8 Å, β = 106.7°, and diffracted to 1.8 Å resolution.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2013; 69(Pt 9):1052-9. DOI:10.1107/S1744309113021490 · 0.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The CD2AP and CIN85 adaptor proteins each employ three SH3 domains to cluster protein partners and ensure efficient signal transduction and downregulation of tyrosine kinase receptors. Using NMR, ITC and SAXS methods, we have characterized several binding modes of the N-terminal SH3 domain (SH3A) of CD2AP and CIN85 against two natural atypical proline-rich ligands from CD2 and Cbl-b and compared and contrasted these data with previous studies and published crystal structures. Our experiments show that the CD2AP-SH3A domain forms a type II dimer with CD2 and both a type I and type II dimeric complex with Cbl-b. Like CD2AP, the CIN85-SH3A domain forms a type II complex with CD2, but a trimeric complex with Cbl-b where the interactions type I and II take place at the same time. Together, these results explain how multiple interactions among similar SH3 domains and ligands can produce a high degree of diversity in tyrosine kinase, cell adhesion or T-cell triggering signaling pathways. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Toxin–antitoxin (TA) modules in bacteria are involved in pathogenesis, antibiotic stress response, persister formation and programmed cell death. The toxin Doc, from the phd/doc module, blocks protein synthesis by targeting the translation machinery. Despite a large wealth of biophysical and biochemical data on the regulatory aspects of the operon transcription and role of Doc co-activator and co-repressor, little is still know on the molecular basis of Doc toxicity. Structural information about this toxin is only available for its inhibited state bound to the antitoxin Phd. Here we report the 1H, 15N and 13C backbone and side chain chemical shift assignments of the toxin Doc from of bacteriophage P1 (the model protein from this family of TA modules) in its free state. The BMRB accession number is 18899.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
In their natural environments, microorganisms compete for space and nutrients, and a major strategy to assist in niche colonization is the deployment of antagonistic compounds directed at competitors, such as secondary metabolites (antibiotics) and antibacterial peptides or proteins (bacteriocins). The latter selectively kill closely related bacteria, which is also the case for members of the LlpA family. Here, we investigate the structure-function relationship for the prototype LlpABW from a saprophytic plant-associated Pseudomonas whose genus-specific target spectrum includes several phytopathogenic pseudomonads. By determining the 3D structure of this protein, we could assign LlpA to the so-called monocot mannose-binding lectin (MMBL) family, representing its first prokaryotic member, and also add a new type of protective function, as the eukaryotic MMBL members have been linked with antiviral, antifungal, nematicidal or insecticidal activities. For the protein containing two similarly folded domains, we constructed site-specific mutants affected in carbohydrate binding and domain chimers from LlpA homologues to show that mannose-specific sugar binding mediated by one domain is required for activity and that the other domain determines target strain specificity. The strategy that evolved for these bacteriocins is reminiscent of the one used by mammalian bactericidal proteins of the RegIII family that recruited a C-type lectin fold to kill bacteria.
[Show abstract][Hide abstract] ABSTRACT: N-Carbamoyl-l-amino acid amidohydrolases (l-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first l-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg(234) in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family.
Journal of bacteriology 08/2012; 194(21):5759-68. DOI:10.1128/JB.01056-12 · 2.81 Impact Factor