•The syndecan ectodomains are flexible random-coil intrinsically disordered proteins.•Their extended conformations have the same size as their cell surface partners.•Their binding-motifs to cells and ...receptors are not accessible in all conformations.•Only some of conformations of the ectodomains may be biologically active.•The syndecan-4 ectodomain forms a disulfide-bonded dimer with extended conformations.
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.
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.
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.
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.
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•Stk2 is a serine/threonine kinase involved in phage defense in Staphylococci•Stk2 is activated by the PacK phage protein•Activation of Stk2 leads to cell death, blocking phage propagation•Stk2 mode of action is similar to some eukaryotic viral defense pathways
Serine/threonine kinases are critical for eukaryotic antiviral responses. Depardieu et al. now report a eukaryotic-like serine/threonine kinase in Staphylococci that protects against bacteriophages by triggering the death of infected cells. This abortive infection system is activated by a phage protein and leads to the extensive phosphorylation of essential cellular pathways.
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 (D
: 11.7 nm, R
: 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.
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