Tailoring the NIR-II Photoluminescence of Single Thiolated Au25 Nanoclusters by Selective Binding to Proteins.

Atomically precise gold nanoclusters are a fascinating class of nanomaterials that exhibit molecule-like properties and have outstanding photoluminescence (PL). Their ultrasmall size, molecular chemistry, and biocompatibility make them extremely appealing for selective biomolecule labeling in investigations of biological mechanisms at the cellular and anatomical levels. In this work, we report a simple route to incorporate a preformed Au25 nanocluster into a model bovine serum albumin (BSA) protein. A new approach combining small-angle X-ray scattering and molecular modeling provides a clear localization of a single Au25 within the protein to a cysteine residue on the gold nanocluster surface. Attaching Au25 to BSA strikingly modifies the PL properties with enhancement and a redshift in the second near-infrared (NIR-II) window. This study paves the way to conrol the design of selective sensitive probes in biomolecules through a ligand-based strategy to enable the optical detection of biomolecules in a cellular environment by live imaging.

Weakening of interaction networks with aging in tip-link protein induces hearing loss.

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among ß-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in ß-strand-rich proteins.

Interdomain linkers tailor the stability of immunoglobulin repeats in polyproteins.

Linkers in polyproteins are considered as mere spacers between two adjacent domains. However, a series of studies using single-molecule force spectroscopy have recently reported distinct thermodynamic stability of I27 in polyproteins with varying linkers and indicated the vital role of linkers in domain stability. A flexible glycine rich linker (-(GGG)n, n ≥ 3) featured unfolding at lower forces than the regularly used arg-ser (RS) based linker. Interdomain interactions among I27 domains in Gly-rich linkers were suggested to lead to reduced domain stability. However, the negative impact of inter domain interactions on domain stability is thermodynamically counter-intuitive and demanded thorough investigations. Here, using an array of ensemble equilibrium experiments and in-silico measurements with I27 singlet and doublets with two aforementioned linkers, we delineate that the inter-domain interactions in fact raise the stability of the polyprotein with RS linker. More surprisingly, a highly flexible Gly-rich linker has no interference on the stability of polyprotein. Overall, we conclude that flexible linkers are preferred in a polyprotein for maintaining domain's independence.

Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss.

Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103-106 pN s-1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

Force-activated catalytic pathway accelerates bacterial adhesion against flow.

Mechanical cues often influence the factors affecting the transition states of catalytic reactions and alter the activation pathway. However, tracking the real-time dynamics of such activation pathways is limited. Using single-molecule trapping of reaction intermediates, we developed a method that enabled us to perform one reaction at one site and simultaneously study the real-time dynamics of the catalytic pathway. Using this, we showed single-molecule calligraphy at nanometer resolution and deciphered the mechanism of the sortase A enzymatic reaction that, counter-intuitively, accelerates bacterial adhesion under shear tension. Our method captured a force-induced dissociation of the enzyme-substrate bond that accelerates the forward reaction 100×, proposing a new mechano-activated catalytic pathway. In corroboration, our molecular dynamics simulations in the presence of force identified a force-induced conformational switch in the enzyme that accelerates proton transfer between CYS184 (acceptor) and HIS120 (donor) catalytic dyads by reducing the inter-residue distances. Overall, the present study opens up the possibility of studying the influence of factors affecting transition states in real time and paves the way for the rational design of enzymes with enhanced efficiency.

Global shapes of F-actin depolymerization-competent minimal gelsolins: insight into the role of g2-g3 linker in pH/Ca2+ insensitivity of the first half.

Because of its ability to rapidly depolymerize F-actin, plasma gelsolin has emerged as a therapeutic molecule in different disease conditions. High amounts of exogenous gelsolin are, however, required to treat animal models of different diseases. Knowing that the F-actin depolymerizing property of gelsolin resides in its N terminus, we made several truncated versions of plasma gelsolin. The smaller versions, particularly the one composed of the first 28-161 residues, depolymerized the F-actin much faster than the native gelsolin and other truncates at the same molar ratios. Although G1-G3 loses its dependence on Ca(2+) or low pH for the actin depolymerization function, interestingly, G1-G2 and its smaller versions were found to regain this requirement. Small angle x-ray scattering-based shape reconstructions revealed that G1-G3 adopts an open shape in both the presence and the absence of Ca(2+) as well as low pH, whereas G1-G2 and residues 28-161 prefer collapsed states in Ca(2+)-free conditions at pH 8. The mutations in the g2-g3 linker resulted in the calcium sensitivity of the mutant G1-G3 for F-actin depolymerization activity, although the F-actin-binding sites remained exposed in the mutant G1-G3 as well as in the smaller truncates even in the Ca(2+)-free conditions at pH 8. Furthermore, unlike wild type G1-G3, calcium-sensitive mutants of G1-G3 acquired closed shapes in the absence of free calcium, implying a role of g2-g3 linker in determining the open F-actin depolymerizing-competent shape of G1-G3 in this condition. We demonstrate that the mobility of the G1 domain, essential for F-actin depolymerization, is indirectly regulated by the gelsolin-like sequence of g2-g3 linker.

Emergence of slip-ideal-slip behavior in tip-links serve as force filters of sound in hearing.

Tip-links in the inner ear convey force from sound and trigger mechanotransduction. Here, we present evidence that tip-links (collectively as heterotetrameric complexes of cadherins) function as force filters during mechanotransduction. Our force-clamp experiments reveal that the tip-link complexes show slip-ideal-slip bond dynamics. At low forces, the lifetime of the tip-link complex drops monotonically, indicating slip-bond dynamics. The ideal bond, rare in nature, is seen in an intermediate force regime where the survival of the complex remains constant over a wide range. At large forces, tip-links follow a slip bond and dissociate entirely to cut-off force transmission. In contrast, the individual tip-links (heterodimers) display slip-catch-slip bonds to the applied forces. While with a phenotypic mutant, we showed the importance of the slip-catch-slip bonds in uninterrupted hearing, our coarse-grained Langevin dynamics simulations demonstrated that the slip-ideal-slip bonds emerge as a collective feature from the slip-catch-slip bonds of individual tip-links.

Visualizing the elusive open shape of G-actin in solution by SAXS data analysis.

Though biochemical data upholds that ATP hydrolysis induces an opening of the nucleotide binding cleft, crystal structures of the G-actin in the absence of profillin represent the closed structure, regardless of the bound ATP/ADP. Analysis of small angle X-ray scattering (SAXS) intensities confirmed that ATP hydrolysis increases the radius of gyration (R(G)) and maximum linear dimension (D(max)) of G-actin molecules from 22.3 to 23.7 Ǻ and 70 to 78 Å, respectively. Kratky analysis confirmed that G-actin molecules behave like globular scattering particles regardless of the bound nucleotide state. Shape reconstruction using dummy residues and inertial axes overlay with known crystal structures confirmed that the ATP or AMP-PNP bound G-actin adopts a compact shape, and the nucleotide binding site opens up with ATP hydrolysis. Importantly, our ADP-state model resembled the open shape seen for ß-actin and hexokinase.

WASCO: A Wasserstein-based Statistical Tool to Compare Conformational Ensembles of Intrinsically Disordered Proteins.

The structural investigation of intrinsically disordered proteins (IDPs) requires ensemble models describing the diversity of the conformational states of the molecule. Due to their probabilistic nature, there is a need for new paradigms that understand and treat IDPs from a purely statistical point of view, considering their conformational ensembles as well-defined probability distributions. In this work, we define a conformational ensemble as an ordered set of probability distributions and provide a suitable metric to detect differences between two given ensembles at the residue level, both locally and globally. The underlying geometry of the conformational space is properly integrated, one ensemble being characterized by a set of probability distributions supported on the three-dimensional Euclidean space (for global-scale comparisons) and on the two-dimensional flat torus (for local-scale comparisons). The inherent uncertainty of the data is also taken into account to provide finer estimations of the differences between ensembles. Additionally, an overall distance between ensembles is defined from the differences at the residue level. We illustrate the potential of the approach with several examples of applications for the comparison of conformational ensembles: (i) produced from molecular dynamics (MD) simulations using different force fields, and (ii) before and after refinement with experimental data. We also show the usefulness of the method to assess the convergence of MD simulations, and discuss other potential applications such as in machine-learning-based approaches. The numerical tool has been implemented in Python through easy-to-use Jupyter Notebooks available at https://gitlab.laas.fr/moma/WASCO.

Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction.

Arrestin-dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR's C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three rhodopsin-like GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR), and the ß2-adernergic receptor (ß2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motifs (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transitions appear to favor arrestin-2 binding. Hence, our results suggest a model in which the phosphorylation-dependent structuration of the GPCR C-terminal regions would modulate arrestin binding and therefore signaling outcomes in arrestin-dependent pathways.

Transient interactions drive the lateral clustering of cadherin-23 on membrane.

Cis and trans-interactions among cadherins secure multicellularity. While the molecular structure of trans-interactions of cadherins is well understood, work to identify the molecular cues that spread the cis-interactions two-dimensionally is still ongoing. Here, we report that transient, weak, yet multivalent, and spatially distributed hydrophobic interactions that are involved in liquid-liquid phase separations of biomolecules in solution, alone can drive the lateral-clustering of cadherin-23 on a membrane. No specific cis-dimer interactions are required for the lateral clustering. In cells, the cis-clustering accelerates cell-cell adhesion and, thus, contributes to cell-adhesion kinetics along with strengthening the junction. Although the physiological connection of cis-clustering with rapid adhesion is yet to be explored, we speculate that the over-expression of cadherin-23 in M2-macrophages may facilitate faster attachments to circulatory tumor cells during metastasis.

Insights into dynamic changes in ADC-7 and P99 cephalosporinases using small angle x-ray scattering (SAXS).

To counter the emergence of ß-lactamase (BL) mediated resistance, design of new ß-lactamase inhibitors (BLIs) is critical. Many high-resolution crystallographic structures of BL complexed with BLIs are available. However, their impact on BLI design is struggling to keep pace with novel and emerging variants. Small angle x-ray scattering (SAXS) in combination with molecular modeling is a useful tool to determine dynamic structures of macromolecules in solution. An important application of SAXS is to determine the conformational changes that occur when BLI bind to BL. To probe if conformational dynamics occur in class C cephalosporinases, we studied SAXS profiles of two clinically relevant class C ß-lactamases, Acinetobacter baumannii ADC-7 and Enterobacter cloacae P99 in apo format complexed with BLIs. Importantly, SAXS data analysis demonstrated that in solution, these representative class C enzymes remain monomeric and did not show the associated assemblies that were seen in various crystal structures. SAXS data acquired for ADC-7 and P99, in apo and inhibitor bound states, clearly showed that these enzymes undergo detectable conformational changes, and these class C ß-lactamases also close upon binding inhibitors as does BlaC. Further analysis revealed that addition of inhibitor led to the compacting of a range of residues around the active site, indicating that the conformational changes that both P99 and ADC-7 undergo are central to inhibitor recognition and efficacy. Our findings support the importance of exploring conformational changes using SAXS analysis in the design of future BLIs.Communicated by Ramaswamy H. Sarma.

Multi-site-specific isotopic labeling accelerates high-resolution structural investigations of pathogenic huntingtin exon-1.

Huntington's disease neurodegeneration occurs when the number of consecutive glutamines in the huntingtin exon-1 (HTTExon1) exceeds a pathological threshold of 35. The sequence homogeneity of HTTExon1 reduces the signal dispersion in NMR spectra, hampering its structural characterization. By simultaneously introducing three isotopically labeled glutamines in a site-specific manner in multiple concatenated samples, 18 glutamines of a pathogenic HTTExon1 with 36 glutamines were unambiguously assigned. Chemical shift analyses indicate the α-helical persistence in the homorepeat and the absence of an emerging toxic conformation around the pathological threshold. Using the same type of samples, the recognition mechanism of Hsc70 molecular chaperone has been investigated, indicating that it binds to the N17 region of HTTExon1, inducing the partial unfolding of the poly-Q. The proposed strategy facilitates high-resolution structural and functional studies in low-complexity regions.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Visual insight into how low pH alone can induce actin-severing ability in gelsolin under calcium-free conditions.

Gelsolin is a key actin cytoskeleton-modulating protein primarily regulated by calcium and phosphoinositides. In addition, low pH has also been suggested to activate gelsolin in the absence of Ca(2+) ions, although no structural insight on this pathway is available except for a reported decrement in its diffusion coefficient at low pH. We also observed ~1.6-fold decrease in the molecular mobility of recombinant gelsolin when buffer pH was lowered from 9 to 5. Analysis of the small angle x-ray scattering data collected over the same pH range indicated that the radius of gyration and maximum linear dimension of gelsolin molecules increased from 30.3 to 34.1 Å and from 100 to 125 Å, respectively. Models generated for each dataset indicated that similar to the Ca(2+)-induced process, low pH also promotes unwinding of this six-domain protein but only partially. It appeared that pH is able to induce extension of the G1 domain from the rest of the five domains, whereas the Ca(2+)-sensitive latch between G2 and G6 domains remains closed. Interestingly, increasing the free Ca(2+) level to merely ~40 nM, the partially open pH 5 shape "sprung open" to a shape seen earlier for this protein at pH 8 and 1 mm free Ca(2+). Also, pH alone could induce a shape where the g3-g4 linker of gelsolin was open when we truncated the C-tail latch from this protein. Our results provide insight into how under physiological conditions, a drop in pH can fully activate the F-actin-severing shape of gelsolin with micromolar levels of Ca(2+) available.

Disentangling polydisperse biomolecular systems by Chemometrics decomposition of SAS data.

The structural characterization of polydisperse systems consisting of multiple coexisting species or conformations is very challenging or impossible with classical approaches. As a consequence, the structural bases of relevant questions related to protein folding, transient partner recognition, conformational transitions or fibrillation remain poorly understood. Small-Angle Scattering (SAS) techniques structurally probe species present in solution in a population-weighted manner, enabling the inspection of polydisperse systems. However, decomposition of these data to derive the contribution of individual components is not straightforward and requires the acquisition of large SAS datasets and adapted mathematical tools. Here, we present a detailed procedure for the usage of the program COSMiCS for the decomposition of SAS datasets. COSMiCS adapts the popular MCR-ALS chemometrics routine to the specificities of scattering data. Through the use of multiple SAS representations, the appropriate scaling of the data and the possibility to simultaneously decompose multiple orthogonal datasets, COSMiCS efficiently disentangles mixtures and provides species-specific structural and thermodynamic/kinetic information of the process under investigation. Although exemplified for a transient biomolecular interaction, our chemometrics strategy can be applied to many other biological processes that can be straightforwardly probed in last generation SAS beamlines. Indeed, recent experimental setups, including microfluidics and stop-flow devices, coupled to fast-reading detectors can yield large concentration or time-dependent datasets that can be decomposed with COSMiCS. Importantly, as an open-source code, previously known features of the system of interest can be introduced as constraints in the optimization, producing robust solutions for biological systems of increasing complexity.

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.

In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation.

Escherichia coli is a Gram-negative bacterium that colonises the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here, we examine SslE from the commensal E. coli Waksman and BL21 (DE3) strains and the enterotoxigenic H10407 and enteropathogenic E2348/69 strains. We reveal that SslE has a unique and dynamic structure in solution and in response to acidification within mature biofilms it can form a unique aggregate with amyloid-like properties. Furthermore, we show that both SslE monomers and aggregates bind DNA in vitro and co-localise with extracellular DNA (eDNA) in mature biofilms, and SslE aggregates may also associate with cellulose under certain conditions. Our results suggest that interactions between SslE and eDNA are important for biofilm maturation in many E. coli strains and SslE may also be a factor that drives biofilm formation in other SslE-secreting bacteria.

Conformational buffering underlies functional selection in intrinsically disordered protein regions.

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

Structural studies on Mycobacterium tuberculosis HddA enzyme using small angle X-ray scattering and dynamics simulation techniques.

Mycobacterium tuberculosis HddA enzyme phosphorylates the M7P substrate and converts it to M7PP product in GDP-D-α-D-heptose biosynthetic pathway. For structural and functional studies on MtbHddA, we have purified the enzyme, which eluted as a monomer from size exclusion column. Purified MtbHddA had ATPase activity. The SAXS analysis supported globular monomeric scattering profile of MtbHddA in solution. The CD analysis showed that MtbHddA contains 45% α-helix, 18% ß-stands, and 32% random coil structures and showed unfolding temperature (TM) ~ 47.5 °C. The unfolding temperature of MtbHddA is enhanced by 1.78±0.41 °C in ATP+Mg2+ bound state, 2.12±0.41 °C in Mannose bound state and 3.07±0.41 °C in Mannose+ ATP+Mg2+ bound state. The apo and M7P +ATP + Mg2+ complexed models of MtbHddA showed that enzyme adopts a classical GHMP sugar kinase fold with conserved ATP+Mg2+ and M7P binding sites. The dynamics simulation analysis on four MtbHddA models showed that ATP+Mg2+ and M7P binding enhanced the stability of active site conformation of MtbHddA. Our study provides important insights into MtbHddA structure and activity, which can be targeted for therapeutic development against M. tuberculosis.