Interactions between proteins and metal cations are central to biochemical processes and shape protein structures. SilE, an intrinsically disordered protein involved in bacterial silver-resistance, folds into α-helices upon binding Ag⁺ ions. Focusing on the B1 peptide fragment from SilE, we investigate the mechanism of Ag⁺-induced folding with simulations and NMR experiments. We first derive force-field parameters for Ag⁺-protein interactions using DFT. Then, we use replica-exchange simulations, deep learning and NMR to map B1’s folding landscape and reveal how Ag⁺ shapes it. Specifically, Ag⁺ binding promotes folding by entropic penalizing the disordered state and electrostatic stabilization of the folded state. We also describe how Ag+ alters the folding pathway. Overall, we improve the understanding of metal-induced protein folding and lay the groundwork for further computational investigations of the bacterial silver-resistance machinery.

The emergence of very high NMR magnetic fields will certainly encourage the study of larger biological systems with their dynamics and interactions. NMR spin relaxation allows probing the dynamical properties of proteins where the ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates in addition to the ¹H–¹⁵N heteronuclear NOE describe the ps–ns timescale. Their analytical representation involves the chemical shift anisotropy (CSA) effect that represents the major contribution at a very high magnetic field above 18.8 T. An accurate analysis of the latter parameters in terms of model free (MF) requires considering its effect. Until now, a uniform value of −160 ppm for the CSA has been widely used to derive the backbone order parameters (S²), giving rise to a large fluctuation of its value at very high magnetic fields. Conversely, the use of a site-specific CSA improves the accurate analysis of protein dynamics but requires a cost-effective experimental multi-field approach. In the present paper, we show how the CSA mainly contributes to the relaxation parameters at 28.2 T compared to lower magnetic fields and may bias the determination of S². We propose to replace the time-consuming measurement of spin relaxation at multiple fields by a combination of molecular dynamics (MD) and the measurement of spin relaxation at one very high magnetic field only. We applied this strategy to three well-folded proteins (ubiquitin, GB3 and ribonuclease H) to show that the determined order parameters are in good agreement with the ones obtained by means of experimental data only.

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.

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.

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.

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.

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.

NLRP3 controls the secretion of inflammatory cytokines IL-1β/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.

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.

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.

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.

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.

A new model of the Zn-based complex, [Zn(LH)(Erx)(ErxH)]ClO₄ (sulfadiazine: LH and enrofloxacin ErxH), has been synthesised with two different, but complementary antibiotics (sulfonamide and quinolon) following an easy procedure. To evaluate the synergetic effect of this multicomponent molecule, the mononuclear complexes [Zn(L)₂(H₂O)(NH₃)] and [Zn(Erx)₃] which each include only one of the two antibiotics (sulfadiazine: LH or enrofloxacin ErxH) were synthesized. All three compounds were characterized, including crystal structure determination by single-crystal X-ray diffraction. For all of the complexes, enrofloxacin is coordinated to Zn(II) by pyridinone and one oxygen atom of the carboxylate group in a monodente mode. The antibacterial activity, determined by the minimum inhibitory concentration on specific bacteria, has been studied on free ligands, on the corresponding mononuclear complexes and on our model.

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 a-helical structuring role of His- and Met-rich sequences of SilE, improving our understanding of its function within the Sil system.

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 wellsampled 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.

Using model peptides, each of the nine MX₂H or HX_nM (n = 1, 2) motifs of the silver resistance protein SilE has been shown to coordinate to one Ag⁺ ion by its histidine and methionine residues with K_d in the μM range. This suggests an Ag⁺ buffering role for SilE in the case of high Ag⁺ overload.

Rotational diffusion (D_rot) is a fundamental property of biomolecules that contains information about molecular dimensions and solute–solvent interactions. While ab initio D_rot 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 D_rot in >10 proteins spanning 5–157 kDa. We also adopt a quaternion-based approach that computes D_rot 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 × τ_theor 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 × τ_theor for GB3. Thus, we encourage reporting of axial decompositions D_x, D_y, D_z 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.