Glycosaminoglycans are polysaccharides that occur both at the cell surface and within extracellular matrices. Through their ability to bind to a large array of proteins, almost 500 of which have been identified to date, including most chemokines, these molecules regulate key biologic processes at the cell-tissue interface. To do so, glycosaminoglycans can provide scaffolds to ensure that proteins mediating specific functions will be presented at the correct site and time and can also directly contribute to biologic activities or signaling processes. The binding of chemokines to glycosaminoglycans, which, at the biochemical level, has been mostly studied using heparin, has traditionally been thought of as a mechanism for maintaining haptotactic gradients within tissues along which cells can migrate directionally. Many aspects of chemokine-glycosaminoglycan interactions, however, also suggest that the formation of these complexes could serve additional purposes that go well beyond a simple immobilization process. In addition, progress in glycobiology has revealed that glycosaminoglycan structures, in term of length, sulfation, and epimerization pattern, are specific for cell, tissue, and developmental stage. Glycosaminoglycan regulation and glycosaminoglycan diversity, which cannot be replicated using heparin, thus suggests that these molecules may fine-tune the immune response by selectively recruiting specific chemokines to cell surfaces. In this context, the aim of the present text is to review the chemokine-glycosaminoglycan complexes described to date and provide a critical analysis of the tools, molecules, and strategies that can be used to structurally and functionally investigate the formation of these complexes.

The chemokine receptor, CXC chemokine receptor 4 (CXCR4), is selective for CXC chemokine ligand 12 (CXCL12), is broadly expressed in blood and tissue cells, and is essential during embryogenesis and hematopoiesis. CXCL14 is a homeostatic chemokine with unknown receptor selectivity and preferential expression in peripheral tissues. Here, we demonstrate that CXCL14 synergized with CXCL12 in the induction of chemokine responses in primary human lymphoid cells and cell lines that express CXCR4. Combining subactive concentrations of CXCL12 with 100–300 nM CXCL14 resulted in chemotaxis responses that exceeded maximal responses that were obtained with CXCL12 alone. CXCL14 did not activate CXCR4-expressing cells (i.e., failed to trigger chemotaxis and Ca2+ mobilization, as well as signaling via ERK1/2 and the small GTPase Rac1); however, CXCL14 bound to CXCR4 with high affinity, induced redistribution of cell-surface CXCR4, and enhanced HIV-1 infection by >3-fold. We postulate that CXCL14 is a positive allosteric modulator of CXCR4 that enhances the potency of CXCR4 ligands. Our findings provide new insights that will inform the development of novel therapeutics that target CXCR4 in a range of diseases, including cancer, autoimmunity, and HIV.—Collins, P. J., McCully, M. L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., Cecchinato, V., Laufer, J. M., Purvanov, V., Monneau, Y. R., Lortat-Jacob, H., Legler, D. F., Uguccioni, M., Thelen, M., Piguet, V., Mellado, M., Moser, B. Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics and function of biomolecule systems by NMR. Despite spectacular progress in the field, such studies are still rather limited in number. One of the main obstacles remains the assignment of the methyl resonances, which is labor intensive and error prone. Typically, NOESY crosspeak patterns are manually correlated to the available crystal structure or an in silico template model of the protein. Here, we propose Methyl Assignment by Graphing Inference Construct (MAGIC), an exhaustive search algorithm with no peak network definition requirement. In order to overcome the combinatorial problem, the exhaustive search is performed locally, i.e. for a small number of methyls connected through-space according to experimental 3D methyl NOESY data. The local network approach drastically reduces the search space. Only the best local assignments are combined together to provide the final output. Assignments that match the data with equivalent – or slightly lower - scores are made available to the user for cross-validation by additional experiments such as methyl-amide NOEs. Several NMR datasets for proteins in the 25–50 kDa range were used during development and for performance evaluation against the manually assigned data. We show that the algorithm is robust, reliable and greatly speeds up the methyl assignment task.

Chemokines promote directional cell migration through binding to G-protein-coupled receptors, and as such are involved in a large array of developmental, homeostatic and pathological processes. They also interact with heparan sulfate (HS), the functional consequences of which depend on the respective location of the receptor- and the HS-binding sites, a detail that remains elusive for most chemokines. Here, to set up a biochemical framework to investigate how HS can regulate CXCL13 activity, we solved the solution structure of CXCL13. We showed that it comprises an unusually long and disordered C-terminal domain, appended to a classical chemokine-like structure. Using three independent experimental approaches, we found that it displays a unique association mode to HS, involving two clusters located in the α-helix and the C-terminal domain. Computational approaches were used to analyse the HS sequences preferentially recognized by the protein and gain atomic-level understanding of the CXCL13 dimerization induced upon HS binding. Starting with four sets of 254 HS tetrasaccharides, we identified 25 sequences that bind to CXCL13 monomer, among which a single one bound to CXCL13 dimer with high consistency. Importantly, we found that CXCL13 can be functionally presented to its receptor in a HS-bound form, suggesting that it can promote adhesion-dependent cell migration. Consistently, we designed CXCL13 mutations that preclude interaction with HS without affecting CXCR5-dependent cell signalling, opening the possibility to unambiguously demonstrate the role of HS in the biological function of this chemokine.

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component,d-glucuronic acid (GlcA), intol-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)nsubstrate or a (IdoA-GlcNS)nproduct. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement ofN-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

Background:The yeast histone chaperone Fpr4 harbors a peptidyl-prolyl isomerase domain of the FK506-binding protein (FKBP) family. Results: Catalytic efficiency toward three peptides from histone H3 relates to residues flanking the central proline. Conclusion: Substrate residues C-terminal to the proline dictate isomerase activity by Fpr4. Significance: The findings reveal new molecular details of substrate peptide recognition by the peptidyl-prolyl isomerase domain.

A simple and cost effective method to independently and stereo-specifically incorporate [1H,13C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [1H,13C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [1H,13C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-13C,2,3-2H2,15N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[13CH3]γ2 labeled protein with an optimal isotope incorporation using up to 50% less labeled Thr than the traditional E. coli strain without the need for 2H-glycine to prevent scrambling.

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