Biology subjects

Hub, J. S.

Publications and source records attributed to Hub, J. S..

29 records · Page 2Linked to original sources

bioRxiv2022

Revealing allostery in PTPN11 SH2 domains from MD simulations

Src-homology 2 (SH2) domains are protein interaction domains that bind to specific peptide motifs containing phosphotyrosine. SHP2, a tyrosine phosphatase encoded by PTPN11 gene, which has been emerged as positive or negative modulator in multiple signaling pathways, contains two SH2 domains, respectively called N-SH2 and C-SH2. These domains play a relevant role in regulating SHP2 activity, either by recognizing its binding partners or by blocking its catalytic site. Considering the multiple functions that these domains carry out in SHP2, N-SH2 and C-SH2 represent an interesting case of study. Here, we present a methodology that permits, by means of the principal component analysis (PCA), to study and to rationalize the structures adopted by the SH2 domains, in terms of the conformations of their binding sites. The structures can be distinguished, grouped, classified and reported in a diagram. This approach permits to identify the accessible conformations of the SH2 domains in different binding conditions and to eventually reveal allosteric interactions. The method further reveals that the conformation dynamics of N-SH2 and C- SH2 strongly differ, which likely reflects their distinct functional roles.

biophysics↗

bioRxiv2022

Structure and ensemble refinement against SAXS data: combining MD simulations with Bayesian inference or with the maximum entropy principle

Small-angle X-ray scattering (SAXS) is a powerful method for tracking conformational transitions of proteins or soft-matter complexes in solution. However, the interpretation of the experimental data is challenged by the low spatial resolution and the low information content of the data, which lead to a high risk of overinterpreting the data. Here, we illustrate how SAXS data can be integrated into all-atom molecular dynamics (MD) simulation to derive atomic structures or heterogeneous ensembles that are compatible with the data. Besides providing atomistic insight, the MD simulation adds physico-chemical information, as encoded in the MD force fields, which greatly reduces the risk of overinterpretation. We present an introduction into the theory of SAXS-driven MD simulations as implemented in GROMACS-SWAXS, a modified version of the GROMACS simulation software. We discuss SAXS-driven parallel-replica ensemble refinement with commitment to the maximum entropy principle as well as a Bayesian formulation of SAXS-driven structure refinement. Practical considerations for running and interpreting the simulations are presented. The methods are freely available via GitLab at https://gitlab.com/cbjh/gromacs-swaxs.

biophysics↗

bioRxiv2022

DNA opening during transcription initiation by RNA polymerase II in atomic detail

RNA polymerase II (RNAP II) is a macro-molecular complex that synthesizes RNA by reading the DNA code, a process called transcription. During transcription initiation, RNAP II opens the double-stranded DNA to expose the DNA template to the active site. The molecular interactions driving and controlling the DNA opening are not well understood. We used all-atom molecular dynamics (MD) simulations to obtain a continuous atomistic pathway for the DNA opening process in human RNAP II. To achieve such large-scale and highly nonlinear transition, we steered the MD simulations along a combination of collective variables involving a guided DNA rotation and a set of path collective variables. The simulations reveal extensive interactions of the DNA with three protein loops near the active site, namely the rudder, fork loop 1, and fork loop 2. According to the simulations, these DNA-protein interactions support DNA opening by attacking Watson-Crick hydrogen bonds, and they stabilize the open DNA bubble by the formation of a wide set of DNA-protein salt bridges.

biophysics↗

bioRxiv2021

Free energies of stalk formation in the lipidomics era

Many biological membranes are asymmetric and exhibit complex lipid composition, comprising hundreds of distinct chemical species. Identifying the biological function and advantage of this complexity is a central goal of membrane biology. Here, we study how membrane complexity controls the energetics of the first steps of membrane fusions, that is, the formation of a stalk. We first present a computationally efficient method for simulating thermodynamically reversible pathways of stalk formation at near-atomic resolution. The new method reveals that the inner leaflet of a typical plasma membrane is far more fusogenic than the outer leaflet, which is likely an adaptation to evolutionary pressure. To rationalize these findings by the distinct lipid compositions, we computed ~200 free energies of stalk formation in membranes with different lipid head groups, tail lengths, tail unsaturations, and sterol content. In summary, the simulations reveal a drastic influence of the lipid composition on stalk formation and a comprehensive fusogenicity map of many biologically relevant lipid classes.

biophysics↗

bioRxiv2020

Do the loops in N-SH2 binding cleft truly serve as allosteric switch in SHP2 activation?

The Src homology-2 domain containing phosphatase SHP2 is a critical regulator of signal transduction, being implicated in cell growth and differentiation. Activating mutations cause developmental disorders and act as oncogenic drivers in hematologic cancers. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as the key "allosteric switch" driving SHP2 activation. To test the putative coupling between binding cleft opening and SHP2 activation as assumed by the "allosteric switch" model, we critically reviewed structural data of SHP2 and we used extensive molecular dynamics (MD) simulation and free energy calculations of isolated N-SH2 in solution, SHP2 in solution, and SHP2 in a crystal environment. Our results demonstrate that the binding cleft in N-SH2 is constitutively flexible and open in solution, and that a closed cleft found in certain structures is a consequence of crystal contacts. The degree of opening of the binding cleft has only a negligible effect on the free energy of SHP2 activation. Instead, SHP2 activation is greatly favored by the opening of the central {beta}-sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation.

biophysics↗

bioRxiv2020

A joint reaction coordinate for computing the free energy landscape of pore nucleation and pore expansion in lipid membranes

Topological transitions of membranes, such as pore formation or membrane fusion, play key roles in biology, biotechnology, and in medical applications. Calculating the related free energy landscapes has been complicated by the fact that such processes involve a sequence of transitions along highly distinct directions in conformational space, making it difficult to define good reaction coordinates (RCs) for the overall process. In this study, we present a new RC capable of driving both pore nucleation and pore expansion in lipid membranes. The potential of mean force (PMF) along the RC computed with molecular dynamics (MD) simulations provides a comprehensive view on the free-energy landscape of pore formation, including a barrier for pore nucleation, the size, free energy, and metastability of the open pore, and the energetic cost for further pore expansion against the line tension of the pore rim. We illustrate the RC by quantifying the effects (i) of simulation system size and (ii) of the addition of dimethyl sulfoxide (DMSO) on the free energy landscape of pore formation. PMF calculations along the RC provide mechanistic and energetic understanding of pore formation, hence they will be useful to rationalize the effects of membrane-active peptides, electric fields, and membrane composition on transmembrane pores.

biophysics↗

bioRxiv2020

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

molecular biology↗

bioRxiv2020

Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics simulations of the N-SH2 domain complexed to 12 different peptides, to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position -2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3 and +5 in an apolar groove of the domain, and interaction of residue -2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues -1, +1, +2 and +4. Finally, negatively charged residues at position +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific of the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core {beta}-sheet and the loop that closes the pY binding pocket.

biophysics↗

bioRxiv2020

Ligand-induced conformational transitions in the activation mechanism of SHP2 tyrosine phosphatase

SHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation.

biophysics↗

bioRxiv2019

Disentangling the Effects of Asymmetry, Shape Fluctuations, Disorder, and Atomic Details on SAXS Curves of Soft Matter Complexes

Small-angle X-ray scattering (SAXS) is a widely used experimental technique, providing structural and dynamic insight into soft-matter complexes and biomolecules under near-native conditions. However, interpreting the one-dimensional scattering profiles in terms of three-dimensional structures and ensembles remains challenging, partly because it is poorly understood how structural information is encoded along the measured scattering angle. We combined all-atom SAXS-restrained ensemble simulations, simplified continuum models, and SAXS experiments of a n-dodecyl-{beta}-D-maltoside (DDM) micelle to decipher the effects of model asymmetry, shape fluctuations, atomic disorder, and atomic details on SAXS curves. Upon interpreting the small-angle regime, we find remarkable agreement between (i) a two-component tri-axial ellipsoid model fitted against the data with (ii) a SAXS-refined all-atom ensemble. However, continuum models fail at wider angles, even if they account for shape fluctuations, disorder, and asymmetry of the micelle. We conclude that modelling atomic details is mandatory for explaining SAXS curves at wider angles.

biophysics↗

bioRxiv2018

Merging in-solution X-ray and neutron scattering data allows fine structural analysis of membrane-protein detergent complexes

AbstractIn-solution small angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labelled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. Firstly, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS which has been adapted to predict SANS data. Secondly, the use of MONSA which allows to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/324103_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (67K):\norg.highwire.dtl.DTLVardef@f7e17corg.highwire.dtl.DTLVardef@1f4987dorg.highwire.dtl.DTLVardef@1475c79org.highwire.dtl.DTLVardef@839b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗