Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central ß-sheet.

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central ß-sheet, and that partial unzipping of this ß-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.

Atomistic ensemble of active SHP2 phosphatase.

SHP2 phosphatase plays an important role in regulating several intracellular signaling pathways. Pathogenic mutations of SHP2 cause developmental disorders and are linked to hematological malignancies and cancer. SHP2 comprises two tandemly-arranged SH2 domains, a catalytic PTP domain, and a disordered C-terminal tail. Under physiological, non-stimulating conditions, the catalytic site of PTP is occluded by the N-SH2 domain, so that the basal activity of SHP2 is low. Whereas the autoinhibited structure of SHP2 has been known for two decades, its active, open structure still represents a conundrum. Since the oncogenic mutant SHP2E76K almost completely populates the active, open state, this mutant has been extensively studied as a model for activated SHP2. By molecular dynamics simulations and accurate explicit-solvent SAXS curve predictions, we present the heterogeneous atomistic ensemble of constitutively active SHP2E76K in solution, encompassing a set of conformational arrangements and radii of gyration in agreement with experimental SAXS data.

Experiment-guided molecular simulations define a heterogeneous structural ensemble for the PTPN11 tandem SH2 domains.

SHP2 plays an important role in regulating cellular processes, and its pathogenic mutations cause developmental disorders and are linked to cancer. SHP2 is a multidomain protein, comprising two SH2 domains arranged in tandem, a catalytic PTP domain, and a disordered C-terminal tail. SHP2 is activated upon binding two linked phosphopeptides to its SH2 domains, and the peptide orientation and spacing between binding sites are critical for enzymatic activation. For decades, the tandem SH2 has been extensively studied to identify the relative orientation of the two SH2 domains that most effectively binds effectors. So far, neither crystallography nor experiments in solution have provided conclusive results. Using experiment-guided molecular simulations, we determine the heterogeneous structural ensemble of the tandem SH2 in solution in agreement with experimental data from small-angle X-ray scattering and NMR residual dipolar couplings. In the solution ensemble, N-SH2 adopts different orientations and positions relative to C-SH2. We suggest that the intrinsic structural plasticity of the tandem SH2 allows SHP2 to respond to external stimuli and is essential for its functional activity.

Molecular simulations of DEAH-box helicases reveal control of domain flexibility by ligands: RNA, ATP, ADP, and G-patch proteins.

DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEAH-box helicases is regulated by cofactors called G-patch proteins. We used all-atom molecular dynamics simulations of the helicases Prp43, Prp22, and DHX15 in various liganded states to investigate how RNA, ADP, ATP, or G-patch proteins influence their conformational dynamics. The simulations suggest that apo helicases are highly flexible, whereas binding of RNA renders the helicases more rigid. ATP and ADP control the stability of the RecA1-RecA2 interface, but they have only a smaller effect on domain flexibility in absence of a RecA1-RecA2 interface. Binding of a G-patch protein to DHX15 imposes a more structured conformational ensemble, characterized by more defined relative domain arrangements and by an increased conformational stability of the RNA tunnel. However, the effect of the G-patch protein on domain dynamics is far more subtle as compared to the effects of RNA or ATP/ADP. The simulations characterize DEAH-box helicase as dynamic machines whose conformational ensembles are strongly defined by the presence of RNA, ATP, or ADP and only fine-tuned by the presence of G-patch proteins.

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. This suppression 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 during RNA suppression remains a long-standing open question. Here, 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 comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

The loops of the N-SH2 binding cleft do not 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 ß-sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation.

An allosteric interaction controls 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.

Cooperative Effects of an Antifungal Moiety and DMSO on Pore Formation over Lipid Membranes Revealed by Free Energy Calculations.

Itraconazole is a triazole drug widely used in the treatment of fungal infections, and it is in clinical trials for treatment of several cancers. However, the drug suffers from poor solubility, while experiments have shown that itraconazole delivery in liposome nanocarriers improves both circulation half-life and tissue distribution. The drug release mechanism from the nanocarrier is still unknown, and it depends on several factors including membrane stability against defect formation. In this work, we used molecular dynamics simulations and potential of mean force (PMF) calculations to quantify the influence of itraconazole on pore formation over lipid membranes, and we compared the effect by itraconazole with a pore-stabilizing effect by the organic solvent dimethyl sulfoxide (DMSO). According to the PMFs, both itraconazole and DMSO greatly reduce the free energy of pore formation, by up to ∼20 kJ mol-1. However, whereas large concentrations of itraconazole of 8 mol % (relative to lipid) were required, only small concentrations of a few mole % DMSO (relative to water) were sufficient to stabilize pores. In addition, itraconazole and DMSO facilitate pore formation by different mechanisms. Whereas itraconazole predominantly aids the formation of a partial defect with a locally thinned membrane, DMSO mainly stabilizes a transmembrane water needle by shielding it from the hydrophobic core. Notably, the two distinct mechanisms act cooperatively upon adding both itraconazole and DMSO to the membrane, as revealed by an additional reduction of the pore free energy. Overall, our simulations reveal molecular mechanisms and free energies of membrane pore formation by small molecules. We suggest that the stabilization of a locally thinned membrane as well as the shielding of a transmembrane water needle from the hydrophobic membrane core may be a general mechanism by which amphiphilic molecules facilitate pore formation over lipid membranes at sufficient concentrations.

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. The 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 (MD) 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 positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core ß sheet and the loop that closes the pY binding pocket.

Detection of functional modes in protein dynamics.

Proteins frequently accomplish their biological function by collective atomic motions. Yet the identification of collective motions related to a specific protein function from, e.g., a molecular dynamics trajectory is often non-trivial. Here, we propose a novel technique termed "functional mode analysis" that aims to detect the collective motion that is directly related to a particular protein function. Based on an ensemble of structures, together with an arbitrary "functional quantity" that quantifies the functional state of the protein, the technique detects the collective motion that is maximally correlated to the functional quantity. The functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. In addition, the motion that displays the largest likelihood to induce a substantial change in the functional quantity is estimated from the given protein ensemble. Two different correlation measures are applied: first, the Pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. Detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. The new approach is illustrated using a number of biomolecules, including a polyalanine-helix, T4 lysozyme, Trp-cage, and leucine-binding protein.

Not only enthalpy: large entropy contribution to ion permeation barriers in single-file channels.

The effect of channel length on the barrier for potassium ion permeation through single-file channels has been studied by means of all-atom molecular dynamics simulations. Using series of peptidic gramicidin-like and simplified ring-structured channels, both embedded in model membranes, we obtained two distinct types of behavior: saturation of the central free energy barriers for peptidic channels and a linear increase in simplified ring-structured channels with increasing channel length. The saturation of the central free energy barrier for the peptidic channels occurs at relatively short lengths, and it is correlated with the desolvation from the bulk water. Remarkably, decomposition of free energy barriers into enthalpic and entropic terms reveals an entropic cost for ion permeation. Furthermore, this entropic cost dominates the ion permeation free energy barrier, since the corresponding free energy contribution is higher than the enthalpic barrier. We conclude that the length dependence of the free energy is enthalpy-dominated, but the entropy is the major contribution to the permeation barrier. The decrease in rotational water motion and the reduction of channel mobility are putative origins for the overall entropic penalty.