Impact of conditioning runs on hydrophilic interaction chromatography repeatability and its application as a second dimension in online comprehensive two-dimensional liquid chromatography.

A common separation approach for polar compounds involves coupling reversed-phase liquid chromatography (RPLC) with hydrophilic interaction chromatography (HILIC) in two-dimensional chromatography. The higher proportion of acetonitrile used in the HILIC mobile phase, which enhances mass spectrometry detection, encourages its use in the second dimension. Previous studies demonstrated that the HILIC column can be partially equilibrated within very short timeframes without compromising retention time stability, rendering it suitable in online comprehensive two-dimensional liquid chromatography (LC×LC) setups. In addition, a specific number of conditioning cycles seems necessary to establish stable retention times. Here, the repeatability of HILIC when employed as second dimension in LC×LC was investigated, with a focus on determining the required number of conditioning cycles to achieve repeatable retention times. Various parameters influenced by the LC×LC online modulation system were studied, such as steep gradient slopes up to 8%, and very short equilibration times, less than or equal to dead time, as well as injection volume and solvent, which depend on the first dimension. Finally, the use of HILIC as a second dimension with tailored conditioning runs was applied to the analysis of hyaluronic acid hydrogel digests. The application of an RPLC×HILIC method using five conditioning runs yielded exceptional stability in second-dimension retention times.

The battle for silver binding: How the interplay between the SilE, SilF, and SilB proteins contributes to the silver efflux pump mechanism.

The resistance of gram-negative bacteria to silver ions is mediated by a silver efflux pump, which mainly relies on a tripartite efflux complex SilCBA, a metallochaperone SilF and an intrinsically disordered protein SilE. However, the precise mechanism by which silver ions are extruded from the cell and the different roles of SilB, SilF, and SilE remain poorly understood. To address these questions, we employed nuclear magnetic resonance and mass spectrometry to investigate the interplay between these proteins. We first solved the solution structures of SilF in its free and Ag+-bound forms, and we demonstrated that SilB exhibits two silver binding sites in its N and C termini. Conversely to the homologous Cus system, we determined that SilF and SilB interact without the presence of silver ions and that the rate of silver dissociation is eight times faster when SilF is bound to SilB, indicating the formation of a SilF-Ag-SilB intermediate complex. Finally, we have shown that SilE does not bind to either SilF or SilB, regardless of the presence or absence of silver ions, further corroborating that it merely acts as a regulator that prevents the cell from being overloaded with silver. Collectively, we have provided further insights into protein interactions within the sil system that contribute to bacterial resistance to silver ions.

Structural and dynamical insights into SilE silver binding from combined analytical probes.

Silver has been used for its antimicrobial properties to fight infection for thousands of years. Unfortunately, some Gram-negative bacteria have developed silver resistance causing the death of patients in a burn unit. The genes responsible for silver resistance have been designated as the sil operon. Among the proteins of the sil operon, SilE has been shown to play a key role in bacterial silver resistance. Based on the limited information available, it has been depicted as an intrinsically disordered protein that folds into helices upon silver ion binding. Herein, this work demonstrates that SilE is composed of 4 clearly identified helical segments in the presence of several silver ions. The combination of analytical and biophysical techniques (NMR spectroscopy, CD, SAXS, HRMS, CE-ICP-MS, and IM-MS) reveals that SilE harbors four strong silver binding sites among the eight sites available. We have also further evidenced that SilE does not adopt a globular structure but rather samples a large conformational space from elongated to more compact structures. This particular structural organization facilitates silver binding through much higher accessibility of the involved His and Met residues. These valuable results will advance our current understanding of the role of SilE in the silver efflux pump complex mechanism and will help in the future rational design of inhibitors to fight bacterial silver resistance.

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase.

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), into l-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)n substrate or a (IdoA-GlcNS)n product. 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 of N-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.

Toolkit for NMR Studies of Methyl-Labeled Proteins.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics, and explore mechanistic insights of large biomolecules by solution NMR. Methyls are relatively insensitive to chemical exchange-induced depolarization and provide superior probes of supramolecular interactions and allostery in such systems. In this chapter, we describe our systematic approach and contributions in the areas of sample preparation, data collection, and data analysis that streamline the application of methyl labeling in solution NMR studies of large proteins. We focus our effort on the initial and often onerous task of methyl resonance assignment and here we detail our approaches to simplify the process. We produce new methyl labeling combinations using Escherichia coli auxotrophs, increase speed, sensitivity, and resolution of NOESY experiments by employing 3D SOFAST-NOESY, and assign methyl resonances from raw data with spectral simulation tools and(or) automatically with minimal expert supervision using the MAGIC algorithm.

Automatic methyl assignment in large proteins by the MAGIC algorithm.

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics and function of biomolecules by NMR. Despite spectacular progress in the field, such studies remain 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, 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 to provide the final output. Assignments that match the data with comparable 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.

Solution structure of CXCL13 and heparan sulfate binding show that GAG binding site and cellular signalling rely on distinct domains.

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.

Basic surface features of nuclear FKBPs facilitate chromatin binding.

The nucleoplasmin family of histone chaperones is identified by a pentamer-forming domain and multiple acidic tracts that mediate histone binding and chaperone activity. Within this family, a novel domain organization was recently discovered that consists of an N-terminal nucleoplasmin-like (NPL) domain and a C-terminal FKBP peptidyl-proline isomerase domain. Saccharomyces cerevisiae Fpr4 is one such protein. Here we report that in addition to its known histone prolyl isomerase activities, the Fpr4 FKBP domain binds to nucleosomes and nucleosome arrays in vitro. This ability is mediated by a collection of basic patches that enable the enzyme to stably associate with linker DNA. The interaction of the Fpr4 FKBP with recombinant chromatin complexes condenses nucleosome arrays independently of its catalytic activity. Based on phylogenetic comparisons we propose that the chromatin binding ability of 'basic' FKBPs is shared amongst related orthologues present in fungi, plants, and insects. Thus, a subclass of FKBP prolyl isomerase enzymes is recruited to linker regions of chromatin.

Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

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.

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.

A simple and cost effective method to independently and stereo-specifically incorporate [(1)H,(13)C]-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 [(1)H,(13)C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [(1)H,(13)C]-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-(13)C,2,3-(2)H2,(15)N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[(13)CH3](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 (2)H-glycine to prevent scrambling.

The sweet spot: how GAGs help chemokines guide migrating cells.

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.

Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization.

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

Chemical shift assignments of the catalytic domain from the yeast proline isomerase Fpr4p.

Yeast Fpr4p belongs to the FK506-binding protein (FKBP) class of peptidyl proline isomerases (PPIases), and has been implicated in regulating the cis-trans conversion of proline residues within histone tails. Here we report the (1)H, (13)C and (15)N chemical shift assignments for the bacterially expressed C-terminal PPIase catalytic domain of Fpr4p. Prediction of secondary structure reveals similarity to domains from other members of the FKBP proline isomerases, including yeast Fpr1p and the prototypic PPIase region from human FKBP12.