Extended disorder at the cell surface: The conformational landscape of the ectodomains of syndecans.

Syndecans are membrane proteoglycans regulating extracellular matrix assembly, cell adhesion and signaling. Their ectodomains can be shed from the cell surface, and act as paracrine and autocrine effectors or as competitors of full-length syndecans. We report the first biophysical characterization of the recombinant ectodomains of the four human syndecans using biophysical techniques, and show that they behave like flexible random-coil intrinsically disordered proteins, and adopt several conformation ensembles in solution. We have characterized their conformational landscapes using native mass spectrometry (MS) and ion-mobility MS, and demonstrated that the syndecan ectodomains explore the majority of their conformational landscape, from minor compact, globular-like, conformations to extended ones. We also report that the ectodomain of syndecan-4, corresponding to a natural isoform, is able to dimerize via a disulfide bond. We have generated a three-dimensional model of the C-terminus of this dimer, which supports the dimerization via a disulfide bond. Furthermore, we have mapped the NXIP adhesion motif of syndecans and their sequences involved in the formation of ternary complexes with integrins and growth factor receptors on the major conformations of their ectodomains, and shown that these sequences are not accessible in all the conformations, suggesting that only some of them are biologically active. Lastly, although the syndecan ectodomains have a far lower number of amino acid residues than their membrane partners, their intrinsic disorder and flexibility allow them to adopt extended conformations, which have roughly the same size as the cell surface receptors (e.g., integrins and growth factor receptors) they bind to.

A comparative analysis of secreted protein disulfide isomerases from the tropical co-endemic parasites Schistosoma mansoni and Leishmania major.

The human parasites Schistosoma mansoni and Leishmania major are co-endemic and a major threat to human health. Though displaying different tissue tropisms, they excrete/secrete similar subsets of intracellular proteins that, interacting with the host extracellular matrix (ECM), help the parasites invading the host. We selected one of the most abundant proteins found in the secretomes of both parasites, protein disulfide isomerase (PDI), and performed a comparative screening with surface plasmon resonance imaging (SPRi), looking for ECM binding partners. Both PDIs bind heparan sulfate; none of them binds collagens; each of them binds further ECM components, possibly linked to the different tropisms. We investigated by small-angle X-ray scattering both PDIs structures and those of a few complexes with host partners, in order to better understand the differences within this conserved family fold. Furthermore, we highlighted a previously undisclosed moonlighting behaviour of both PDIs, namely a concentration-dependent switch of function from thiol-oxidoreductase to holdase. Finally, we have tried to exploit the differences to look for possible compounds able to interfere with the redox activity of both PDI.

Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners.

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (Dmax: 11.7 nm, Rg: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway.

A Eukaryotic-like Serine/Threonine Kinase Protects Staphylococci against Phages.

Organisms from all domains of life are infected by viruses. In eukaryotes, serine/threonine kinases play a central role in antiviral response. Bacteria, however, are not commonly known to use protein phosphorylation as part of their defense against phages. Here we identify Stk2, a staphylococcal serine/threonine kinase that provides efficient immunity against bacteriophages by inducing abortive infection. A phage protein of unknown function activates the Stk2 kinase. This leads to the Stk2-dependent phosphorylation of several proteins involved in translation, global transcription control, cell-cycle control, stress response, DNA topology, DNA repair, and central metabolism. Bacterial host cells die as a consequence of Stk2 activation, thereby preventing propagation of the phage to the rest of the bacterial population. Our work shows that mechanisms of viral defense that rely on protein phosphorylation constitute a conserved antiviral strategy across multiple domains of life.

Comparative analysis of Leishmania exoproteomes: implication for host-pathogen interactions.

Leishmaniasis is a vector-borne disease caused by the protozoa Leishmania. We have analyzed and compared the sequences of three experimental exoproteomes of Leishmania promastigotes from different species to determine their specific features and to identify new candidate proteins involved in interactions of Leishmania with the host. The exoproteomes differ from the proteomes by a decrease in the average molecular weight per protein, in disordered amino acid residues and in basic proteins. The exoproteome of the visceral species is significantly enriched in sites predicted to be phosphorylated as well as in features frequently associated with molecular interactions (intrinsic disorder, number of disordered binding regions per protein, interaction and/or trafficking motifs) compared to the other species. The visceral species might thus have a larger interaction repertoire with the host than the other species. Less than 10% of the exoproteomes contain heparin-binding and RGD sequences, and ~30% the host targeting signal RXLXE/D/Q. These latter proteins might thus be exported inside the host cell during the intracellular stage of the infection. Furthermore we have identified nine protein families conserved in the three exoproteomes with specific combinations of Pfam domains and selected eleven proteins containing at least three interaction and/or trafficking motifs including two splicing factors, phosphomannomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, the paraflagellar rod protein-1D and a putative helicase. Their role in host-Leishmania interactions warrants further investigation but the putative ATP-dependent DEAD/H RNA helicase, which contains numerous interaction motifs, a host targeting signal and two disordered regions, is a very promising candidate.

The extended conformation of the 2.9-Å crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus.

PASTA (penicillin-binding protein and serine/threonine kinase associated) modules are found in penicillin-binding proteins and bacterial serine/threonine kinases mainly from Gram-positive Firmicutes and Actinobacteria. They may act as extracellular sensors by binding peptidoglycan fragments. We report here the first crystal structure of a multiple-PASTA domain from Ser/Thr kinase, that of the protein serine/threonine kinase 1 (Stk1) from the Firmicute Staphylococcus aureus. The extended conformation of the three PASTA subunits differs strongly from the compact conformation observed in the two-PASTA domain of penicillin-binding protein PBP2x, whereas linear conformations were also reported for two-subunit fragments of the four-PASTA domain of the Actinobacteria Mycobacterium tuberculosis studied by liquid NMR. Thus, a stretched organization appears to be the signature of modular PASTA domains in Ser/Thr kinases. Signal transduction to the kinase domain is supposed to occur via dimerization and ligand binding. A conserved X-shaped crystallographic dimer stabilized by intermolecular interactions between the second PASTA subunits of each monomer is observed in the two crystal forms of Stk1 that we managed to crystallize. Extracellular PASTA domains are composed of at least two subunits, and this molecular assembly is a plausible candidate for the biological dimer. We have also performed docking experiments, which predict that the hinge regions of the PASTA domain can accommodate peptidoglycan. Finally, a three-dimensional homology molecular model of full-length Stk1 was generated, suggesting an interaction between the kinase domain and the cytoplasmic face of the plasma membrane via a eukaryotic-like juxtamembrane domain. A comprehensive activation mechanism for bacterial Ser/Thr kinases is proposed with the support of these structural data.

The Staphylococcus aureus autoinducer-2 synthase LuxS is regulated by Ser/Thr phosphorylation.

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.

Phosphorylation of the virulence regulator SarA modulates its ability to bind DNA in Staphylococcus aureus.

Staphylococcus aureus is one of the main bacterial species of clinical importance. Its virulence is considered multifactorial and is attributed to the combined action of a variety of molecular determinants including the virulence regulator SarA. Phosphorylation of SarA was observed to occur in vivo. From this finding, SarA was overproduced and purified to homogeneity. In an in vitro assay, it was found to be unable to autophosphorylate, but was effectively modified at threonine and serine residues by each of the two Ser/Thr kinases of S. aureus, Stk1 (PknB) and SA0077, respectively. In addition, phosphorylation of SarA was shown to modify its ability to bind DNA. Together, these data support the concept that protein phosphorylation directly participates, at the transcription level, in the control of bacterial pathogenicity.

Crystallization and initial X-ray diffraction study of the three PASTA domains of the Ser/Thr kinase Stk1 from the human pathogen Staphylococcus aureus.

PASTA subunits (approximately 70 amino acids) are specific to bacterial serine/threonine kinases and to penicillin-binding proteins (PBPs) and are involved in the synthesis of peptidoglycan. The human pathogen Staphylococcus aureus contains a serine/threonine kinase, Stk1, which plays a major role in virulence. A recombinant His-tagged portion of the extracellular domain of Stk1 containing three PASTA subunits has been crystallized using zinc sulfate as a crystallizing agent. The crystals belonged to the tetragonal space group P4(1)22, with unit-cell parameters a = 68.0, b = 68.0, c = 158.1 angstrom. Structure determination by the MAD method is now in progress.

Characterization of a serine/threonine kinase involved in virulence of Staphylococcus aureus.

Staphylococcus aureus is a common human cutaneous and nasal commensal and a major life-threatening pathogen. Adaptation to the different environments encountered inside and outside the host is a crucial requirement for survival and colonization. We identified and characterized a eukaryotic-like serine/threonine kinase with three predicted extracellular PASTA domains (SA1063, or Stk1) and its associated phosphatase (SA1062, or Stp1) in S. aureus. Biochemical analyses revealed that Stk1 displays autokinase activity on threonine and serine residues and is localized to the membrane. Stp1 is a cytoplasmic protein with manganese-dependent phosphatase activity toward phosphorylated Stk1. In-frame deletions of the stk1 and stp1 genes were constructed in S. aureus strain 8325-4. Phenotypic analyses of the mutants revealed reduced growth of the stk1 mutant in RPMI 1640 defined medium that was restored when adenine was added to the medium. Furthermore, the stk1 mutant displayed increased resistance to Triton X-100 and to fosfomycin, suggesting modifications in cell wall metabolism. The stk1 mutant was tested for virulence in a mouse pyelonephritis model and found to be strongly reduced for survival in the kidneys (approximately 2-log-unit decrease) compared to the parental strain. Renal histopathological analyses showed severe inflammatory lesions in mice infected with the parental S. aureus SH1000 strain, whereas the Deltastk1 mutant led to only minimal renal lesions. These results confirm the important role of Stk1 for full expression of S. aureus pathogenesis and suggest that phosphorylation levels controlled by stk1 are essential in controlling bacterial survival within the host.

A modified His-tag vector for the production of recombinant protein kinases.

Histidine-tag (His-tag) is the most frequently used tag to label and purify recombinant protein kinases, namely autokinases. However, when analyzing protein phosphorylation, it appears that this modification occurs not only on the kinase itself but also on several serine residues present in the vector-derived His-tag sequence. These parasite modifications can thus lead to misinterpretation of the data concerning protein phosphorylation. We report here on a modified vector devoid of serine residues in the tag and, therefore, more appropriate and secure for studying protein phosphorylation.

Several enzymes of the central metabolism are phosphorylated in Staphylococcus aureus.

Staphylococcus aureus is an important human and animal pathogen that harbors protein kinases. The proteins phosphorylated in this bacterium grown on glucose minimal medium have been in vivo labeled with[(32)P]-orthophosphate and analyzed by two-dimensional gel electrophoresis followed by MS. A total of 11 glycolytic phosphoproteins have been identified and verified. In vitro analyses have revealed that phosphorylation of these glycolytic enzymes is catalysed primarily through the activity of an endogenous serine/threonine kinase and to a lesser extent by a tyrosine kinase. The identification of these phosphoproteins should prove helpful in understanding and unravelling of the role of phosphorylation with respect to pathogenesis and virulence in this organism.

Staphylococcus aureus operates protein-tyrosine phosphorylation through a specific mechanism.

Protein phosphorylation on tyrosine has been originally characterized in animal systems and has been shown to be involved in several fundamental processes including signal transduction, growth control, and malignancy. It has been later demonstrated to occur also in a number of bacteria, and recent data suggest that it may participate in the control of bacterial pathogenicity. In this work, we provide evidence that the gram-positive human pathogen Staphylococcus aureus harbors a protein-tyrosine kinase activity. This activity is borne by a protein, termed Cap5B2, whose phosphorylating capacity is expressed only in the presence of a stimulatory protein, either Cap5A1 or Cap5A2, that enhances its affinity for the phosphoryl donor ATP. In fact, the last 27/29 amino acids of the C-terminal domain of either polypeptide are sufficient for stimulating Cap5B2 activity. The stimulation of Cap5B2 by Cap5A1 involves essentially three amino acid residues in a helix of Cap5A1 (Asp202, Glu203, and Asp205) and three residues in a helix (helix 7) of Cap5B2 (Glu190, Lys192, and Lys193), thus suggesting helix-helix interaction between these two proteins. This type of helix-helix interaction resembles the interaction required for the activation of MinD ATPase by MinE protein in the process of septum-site determination, MinD sharing sequence similarity with Cap5B2. Such activation mechanism is described here in a gram-positive bacterial tyrosine kinase, and differs from the activation mechanism previously proposed for gram-negative bacteria. Therefore, it appears that S. aureus, and possibly other gram-positive bacteria, utilizes a specific molecular mechanism for triggering protein-tyrosine kinase activity.

Autophosphorylation of the 16 kDa and 70 kDa antigens (Hsp 16.3 and Hsp 70) of Mycobacterium tuberculosis.

Several antigens of Mycobacterium tuberculosis, identified by monoclonal antibodies, have been previously cloned and are being exploited in the development of improved vaccines and diagnostic reagents. In this study, the molecular characteristics of two of these antigens, the immunodominant proteins Hsp 16.3 and Hsp 70, were analysed in further detail by assessing their capacity to undergo protein phosphorylation, a chemical modification frequently used by organisms to adjust to environmental variations. Hsp 16.3 was overproduced in an Escherichia coli expression system and purified to homogeneity. Upon incubation in the presence of radioactive ATP, it was shown to possess autophosphorylation activity. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at serine residues. In addition, cross-linking experiments demonstrated that it could tightly bind to ATP. Purified Hsp 70 was also shown to autophosphorylate but phosphorylation occurred exclusively at threonine residues. This reaction was found to be strongly stimulated by calcium ions. These data indicate that both structural and functional similarities exist between Hsp 16.3 (Acr) and alpha-crystallin, a eukaryotic protein which plays an important role in maintaining the transparency of the vertebrate eye, and that the functional properties of Hsp 70 from M. tuberculosis are similar to those of other bacterial members of the Hsp 70 family, particularly the E. coli homologue DnaK.

Protein phosphorylation on tyrosine in bacteria.

Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.

Staphylococcus aureus contains two low-molecular-mass phosphotyrosine protein phosphatases.

The analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacterium Staphylococcus aureus suggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria. To check this prediction, the corresponding genes were cloned and overexpressed in an Escherichia coli system. Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties. As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40 degrees C. In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors, N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases.