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.

Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence.

The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system (T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island (FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique α-helical N-terminal extension and a domain of unknown function (DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal α-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF.

Funnel-metadynamics and solution NMR to estimate protein-ligand affinities.

One of the intrinsic properties of proteins is their capacity to interact selectively with other molecules in their environment, inducing many chemical equilibria each differentiated by the mutual affinities of the components. A comprehensive understanding of these molecular binding processes at atomistic resolution requires formally the complete description of the system dynamics and statistics at the relevant time scales. While solution NMR observables are averaged over different time scales, from picosecond to second, recent new molecular dynamics protocols accelerated considerably the simulation time of realistic model systems. Based on known ligands recently discovered either by crystallography or NMR for the human peroxiredoxin 5, their affinities were for the first time accurately evaluated at atomistic resolution comparing absolute binding free-energy estimated by funnel-metadynamics simulations and solution NMR experiments. In particular, free-energy calculations are demonstrated to discriminate two closely related ligands as pyrocatechol and 4-methylpyrocathecol separated just by 1 kcal/mol in aqueous solution. The results provide a new experimental and theoretical basis for the estimation of ligand-protein affinities.

Evidence for cooperative and domain-specific binding of the signal transducing adaptor molecule 2 (STAM2) to Lys63-linked diubiquitin.

As the upstream component of the ESCRT (endosomal sorting complexes required for transport) machinery, the ESCRT-0 complex is responsible for directing ubiquitinated membrane proteins to the multivesicular body pathway. ESCRT-0 is formed by two subunits known as Hrs (hepatocyte growth factor-regulated substrate) and STAM (signal transducing adaptor molecule), both of which harbor multiple ubiquitin-binding domains (UBDs). In particular, STAM2 possesses two UBDs, the VHS (Vps27/Hrs/Stam) and UIM (ubiquitin interacting motif) domains, connected by a 20-amino acid flexible linker. In the present study, we report the interactions of the UIM domain and VHS-UIM construct of STAM2 with monoubiquitin (Ub), Lys(48)- and Lys(63)-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin, Lys(48)- and Lys(63)-linked diubiquitins with the same affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys(63)-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys(63)-linked diubiquitin stabilizes the helical structure of the UIM domain and that the corresponding complex adopts a specific structural organization responsible for its greater affinity. In contrast, binding of VHS-UIM to Lys(48)-linked diubiquitin and monoubiquitin is not cooperative and does not show any avidity. These results may explain the better sorting efficiency of some cargoes polyubiquitinated with Lys(63)-linked chains over monoubiquitinated cargoes or those tagged with Lys(48)-linked chains.

Revisiting Schistosoma mansoni Micro-Exon Gene (MEG) Protein Family: A Tour into Conserved Motifs and Annotation.

Genome sequencing of the human parasite Schistosoma mansoni revealed an interesting gene superfamily, called micro-exon gene (meg), that encodes secreted MEG proteins. The genes are composed of short exons (3-81 base pairs) regularly interspersed with long introns (up to 5 kbp). This article recollects 35 S. mansoni specific meg genes that are distributed over 7 autosomes and one pair of sex chromosomes and that code for at least 87 verified MEG proteins. We used various bioinformatics tools to produce an optimal alignment and propose a phylogenetic analysis. This work highlighted intriguing conserved patterns/motifs in the sequences of the highly variable MEG proteins. Based on the analyses, we were able to classify the verified MEG proteins into two subfamilies and to hypothesize their duplication and colonization of all the chromosomes. Together with motif identification, we also proposed to revisit MEGs' common names and annotation in order to avoid duplication, to help the reproducibility of research results and to avoid possible misunderstandings.

Divide, conquer and reconstruct: How to solve the 3D structure of recalcitrant Micro-Exon Gene (MEG) protein from Schistosoma mansoni.

Micro-Exon Genes are a widespread class of genes known for their high variability, widespread in the genome of parasitic trematodes such as Schistosoma mansoni. In this study, we present a strategy that allowed us to solve the structures of three alternatively spliced isoforms from the Schistoma mansoni MEG 2.1 family for the first time. All isoforms are hydrophobic, intrinsically disordered, and recalcitrant to be expressed in high yield in heterologous hosts. We resorted to the chemical synthesis of shorter pieces, before reconstructing the entire sequence. Here, we show that isoform 1 partially folds in a-helix in the presence of trifluoroethanol while isoform 2 features two rigid elbows, that maintain the peptide as disordered, preventing any structuring. Finally, isoform 3 is dominated by the signal peptide, which folds into a-helix. We demonstrated that combining biophysical techniques, like circular dichroism and nuclear magnetic resonance at natural abundance, with in silico molecular dynamics simulation for isoform 1 only, was the key to solve the structure of MEG 2.1. Our results provide a crucial piece to the puzzle of this elusive and highly variable class of proteins.

NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin.

The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin. We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS for K48- and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin.

NMR reveals the interplay between SilE and SilB model peptides in the context of silver resistance.

SilE and SilB are both proteins involved in the silver efflux pump found in Gram-negative bacteria such as S. typhimurium. Using model peptides along with NMR and CD experiments, we show how SilE may store silver ions prior to delivery and we hypothesize for the first time the interplay between SilB and SilE.

NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly.

NLRP3 controls the secretion of inflammatory cytokines IL-1ß/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.

Accurate Prediction of Protein NMR Spin Relaxation by Means of Polarizable Force Fields. Application to Strongly Anisotropic Rotational Diffusion.

Among the various biophysical methods available to investigate protein dynamics, NMR presents the ability to scrutinize protein motions on a broad range of time scales. 1H-15N NMR spin relaxation experiments can reveal the extent of protein motions across the picosecond-nanosecond dynamics probed by the fundamental parameters 15N-R1, 15N-R2, and 1H-15N NOE that can be well sampled by molecular dynamics (MD) simulations. An accurate prediction of these parameters is subjected to a proper description of the rotational diffusion and anisotropy. Indeed, a strong rotational anisotropy has a profound effect on the various relaxation parameters and could be mistaken for conformational exchange. Although the principle of NMR spin relaxation predictions from MD is now well established, numerous NMR/MD comparisons have hitherto focused on proteins that show low to moderate anisotropy and make use of a scaling factor to remove artifacts arising from water model-dependence of the rotational diffusion. In the present work, we have used NMR to characterize the rotational diffusion of the α-helical STAM2-UIM domain by measuring the 15N-R1, 15N-R2, and 1H-15N NOE relaxation parameters. We therefore highlight the use of the polarizable AMOEBA force field (FF) and show that it improves the prediction of the rotational diffusion in the particular case of strong rotational anisotropy, which in turn enhances the prediction of the 15N-R1, 15N-R2, and 1H-15N NOE relaxation parameters without the requirement of a scaling factor. Our findings suggest that the use of polarizable FFs could potentially enrich our understanding of protein dynamics in situations where charge distribution or protein shape is remodeled over time like in the case of multidomain proteins or intrinsically disordered proteins.

Molecular recognition of ubiquitin and Lys63-linked diubiquitin by STAM2 UIM-SH3 dual domain: the effect of its linker length and flexibility.

Multidomain proteins represent a broad spectrum of the protein landscape and are involved in various interactions. They could be considered as modular building blocks assembled in distinct fashion and connected by linkers of varying lengths and sequences. Due to their intrinsic flexibility, these linkers provide proteins a subtle way to modulate interactions and explore a wide range of conformational space. In the present study, we are seeking to understand the effect of the flexibility and dynamics of the linker involved in the STAM2 UIM-SH3 dual domain protein with respect to molecular recognition. We have engineered several constructs of UIM-SH3 with different length linkers or domain deletion. By means of SAXS and NMR experiments, we have shown that the modification of the linker modifies the flexibility and the dynamics of UIM-SH3. Indeed, the global tumbling of both the UIM and SH3 domain is different but not independent from each other while the length of the linker has an impact on the ps-ns time scale dynamics of the respective domains. Finally, the modification of the flexibility and dynamics of the linker has a drastic effect on the interaction of UIM-SH3 with Lys63-linked diubiquitin with a roughly eight-time weaker dissociation constant.

Regulation of measles virus gene expression by P protein coiled-coil properties.

The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the "a" or "d" hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.

Ab Initio Prediction of NMR Spin Relaxation Parameters from Molecular Dynamics Simulations.

1H-15N NMR spin relaxation and relaxation dispersion experiments can reveal the time scale and extent of protein motions across the ps-ms range, where the ps-ns dynamics revealed by fundamental quantities R1, R2, and heteronuclear NOE can be well-sampled by molecular dynamics simulations (MD). Although the principles of relaxation prediction from simulations are well-established, numerous NMR-MD comparisons have hitherto focused upon the aspect of order parameters S2 due to common artifacts in the prediction of transient dynamics. We therefore summarize here all necessary components and highlight existing and proposed solutions, such as the inclusion of quantum mechanical zero-point vibrational corrections and separate MD convergence of global and local motions in coarse-grained and all-atom force fields, respectively. For the accuracy of the MD prediction to be tested, two model proteins GB3 and Ubiquitin are used to validate five atomistic force fields against published NMR data supplemented by the coarse-grained force field MARTINI+EN. In Amber and CHARMM-type force fields, quantitative agreement was achieved for structured elements with minimum adjustment of global parameters. Deviations from experiment occur in flexible loops and termini, indicating differences in both the extent and time scale of backbone motions. The lack of systematic patterns and water model dependence suggests that modeling of the local environment limits prediction accuracy. Nevertheless, qualitative accuracy in a 2 µs CHARMM36m Stam2 VHS domain simulation demonstrates the potential of MD-based interpretation in combination with NMR-measured dynamics, increasing the utility of spin relaxation in integrative structural biology.

Alpha-helical folding of SilE models upon Ag(His)(Met) motif formation.

The SilE protein is suspected to have a prominent role in Ag+ detoxification of silver resistant bacteria. Using model peptides, we elucidated both qualitative and quantitative aspects of the Ag+-induced α-helical structuring role of His- and Met-rich sequences of SilE, improving our understanding of its function within the Sil system.

NMR screening applied to the fragment-based generation of inhibitors of creatine kinase exploiting a new interaction proximate to the ATP binding site.

Using an in-house fragment NMR library, we identified a set of ligands that bind rabbit muscular creatine kinase, an enzyme involved in key ATP-dependent processes. The ligands docked to the crystal structures of creatine kinase indicated that a phenylfuroic acid could enter into a pocket adjacent to the nucleotide binding site. This fragment served as an anchor to develop in silico a series of potential inhibitors which could partly access the nucleotide binding site. The short synthesis of only four derivatives provided entirely novel hit compounds that reversibly inhibit creatine kinase at micromolar concentrations with a mixed ATP-competitive/noncompetitive mechanism in agreement with the structural model of the inhibited enzyme. These initial biologically active compounds are novel and modular and exploit a new interaction proximate to the ATP binding site.

Computing the Rotational Diffusion of Biomolecules via Molecular Dynamics Simulation and Quaternion Orientations.

Rotational diffusion (Drot) is a fundamental property of biomolecules that contains information about molecular dimensions and solute-solvent interactions. While ab initio Drot prediction can be achieved by explicit all-atom molecular dynamics simulations, this is hindered by both computational expense and limitations in water models. We propose coarse-grained force fields as a complementary solution, and show that the MARTINI force field with elastic networks is sufficient to compute Drot in >10 proteins spanning 5-157 kDa. We also adopt a quaternion-based approach that computes Drot orientation directly from autocorrelations of best-fit rotations as used in, e.g., RMSD algorithms. Over 2 µs trajectories, isotropic MARTINI+EN tumbling replicates experimental values to within 10-20%, with convergence analyses suggesting a minimum sampling of >50 × τtheor to achieve sufficient precision. Transient fluctuations in anisotropic tumbling cause decreased precision in predictions of axisymmetric anisotropy and rhombicity, the latter of which cannot be precisely evaluated within 2000 × τtheor for GB3. Thus, we encourage reporting of axial decompositions Dx, Dy, Dz to ease comparability between experiment and simulation. Where protein disorder is absent, we observe close replication of MARTINI+EN Drot orientations versus CHARMM22*/TIP3p and experimental data. This work anticipates the ab initio prediction of NMR-relaxation by combining coarse-grained global motions with all-atom local motions.

Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway.

The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface patch and the basic/polar residues surrounding it. Ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin receptor and shows strong preference for K48 linkages over K11 and K63. Furthermore, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S proteasome. Finally, ubistatin B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface.

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

NMR Reveals the Interplay among the AMSH SH3 Binding Motif, STAM2, and Lys63-Linked Diubiquitin.

AMSH [associated molecule with a Src homology 3 domain of signal transducing adaptor molecule (STAM)] is one of the deubiquitinating enzymes associated in the regulation of endocytic cargo trafficking. It shows an exquisite selectivity for Lys63-linked polyubiquitin chains that are the main chains involved in cargo sorting. The first step requires the ESCRT-0 complex that comprises the STAM and hepatocyte growth factor-regulated substrate (Hrs) proteins. Previous studies have shown that the presence of the STAM protein increases the efficiency of Lys63-linked polyubiquitin chain cleavage by AMSH, one of the deubiquitinating enzyme involved in lysosomal degradation. In the present study, we are seeking to understand if a particular structural organization among these three key players is responsible for the stimulation of the catalytic activity of AMSH. To address this question, we first monitored the interaction between the ubiquitin interacting motif (UIM)-SH3 construct of STAM2 and the Lys63-linked diubiquitin (Lys63-Ub2) chains by means of NMR. We show that Lys63-Ub2 is able to bind either the UIM or the SH3 domain without any selectivity. We further demonstrate that the SH3 binding motif (SBM) of AMSH (AMSH-SBM) outcompetes Lys63-Ub2 for binding SH3. Additionally, we show how different AMSH-SBM variants, modified by their sequence and length, exhibit similar equilibrium dissociation constants when binding SH3 but significantly differ in their dissociation rate constants. Finally, we report the solution NMR structure of the AMSH-SBM/SH3 complex and propose a structural organization where the AMSH-SBM interacts with the STAM2-SH3 domain and contributes to the correct positioning of AMSH prior to polyubiquitin chains' cleavage.