Allostery Frustrates the Experimentalist

Gianni, Stefano; Jemth, Per

doi:10.1016/j.jmb.2022.167934

Cited by 14 publications

(10 citation statements)

References 64 publications

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“…This aspect suggests that such residues, which are not directly involved in the function of the protein, can still modulate binding through the intramolecular energetic network that may be at the basis of the ability of C‐SH2 to accommodate different partners to its binding pocket in the intracellular environment. However, although these ‘allosteric’ sites may represent possible targets for pharmacological strategies aimed to modulate the binding properties of protein–protein interaction domains, the functional relevance of the energetic networks in which they are involved is currently the subject of debate (Gianni & Jemth, 2023 ). Moreover, the analysis of thermodynamic parameters allowed us to highlight the role of V646 residue of Gab2 in the binding with C‐SH2, pinpointing a dual contribution from electrostatics and hydrophobic interactions in the early and late events of binding with the ligand.…”

Section: Discussionmentioning

confidence: 99%

An intramolecular energetic network regulates ligand recognition in a SH2 domain

et al. 2023

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In an effort to investigate the molecular determinants of ligand recognition of the C‐terminal SH2 domain of the SHP2 protein, we conducted extensive site‐directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in‐depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site‐directed variants of C‐SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C‐SH2 binding specificity. Results highlighted non‐optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C‐SH2 domain.

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Section: Discussionmentioning

confidence: 99%

An intramolecular energetic network regulates ligand recognition in a SH2 domain

et al. 2023

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“…Allostery ,, is a fundamental mechanism regulating such functions, whereby distal parts of a protein or multimeric complex (often not intuitively linkable to active sites or binding interfaces) dynamically communicate with each other, with far-reaching repercussions on cellular activities: examples of the many aspects inextricably dependent on allostery include enzyme function, − protein folding by chaperones, − signal transduction, and regulation of transcription and metabolism. Understanding of allostery has been constantly expanding since seminal studies in the 1960s and 1980s, with the topic being extensively reviewed and reformulated. ,− ,− …”

Section: Introductionmentioning

confidence: 99%

“…Computational techniques ,, are essential to obtain an atomistic understanding of the determinants of allosteric regulation: ,− not only is this fundamental to drive the design of better, more “personalized” allosteric drugs and of tailored biocatalysts but also, e.g. , , in the field of molecular diagnostics.…”

Section: Introductionmentioning

confidence: 99%

Decrypting Allostery in Membrane-Bound K-Ras4B Using Complementary In Silico Approaches Based on Unbiased Molecular Dynamics Simulations

Castelli,

Marchetti,

Osuna

et al. 2023

J. Am. Chem. Soc.

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Protein functions are dynamically regulated by allostery, which enables conformational communication even between faraway residues, and expresses itself in many forms, akin to different "languages": allosteric control pathways predominating in an unperturbed protein are often unintuitively reshaped whenever biochemical perturbations arise (e.g., mutations). To accurately model allostery, unbiased molecular dynamics (MD) simulations require integration with a reliable method able to, e.g., detect incipient allosteric changes or likely perturbation pathways; this is because allostery can operate at longer time scales than those accessible by plain MD. Such methods are typically applied singularly, but we here argue their joint application�as a "multilingual" approach�could work significantly better. We successfully prove this through unbiased MD simulations (∼100 μs) of the widely studied, allosterically active oncotarget K-Ras4B, solvated and embedded in a phospholipid membrane, from which we decrypt allostery using four showcase "languages": Distance Fluctuation analysis and the Shortest Path Map capture allosteric hotspots at equilibrium; Anisotropic Thermal Diff usion and Dynamical Non-Equilibrium MD simulations assess perturbations upon, respectively, either superheating or hydrolyzing the GTP that oncogenically activates K-Ras4B. Chosen "languages" work synergistically, providing an articulate, mutually coherent, experimentally consistent picture of K-Ras4B allostery, whereby distinct traits emerge at equilibrium and upon GTP cleavage. At equilibrium, combined evidence confirms prominent allosteric communication from the membrane-embedded hypervariable region, through a hub comprising helix α5 and sheet β5, and up to the active site, encompassing allosteric "switches" I and II (marginally), and two proposed pockets. Upon GTP cleavage, allosteric perturbations mostly accumulate on the switches and documented interfaces.

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“…Understanding of allostery has been constantly expanding since seminal studies in the 1960s 18 and 1980s, 19 with the topic being extensively reviewed and reformulated. 4,[7][8][9][10][20][21][22][23][24] Dynamic (residue-mediated) allosteric communication pathways arise even in the more conformationally constrained proteins, [6][7][8]19 mediating the transition between distinct energy-wells in biomolecules characterized by well-defined "folding funnel" minima. 5,10,23,25 Such minima associated with the native structure can be rugged and different sub-states can exist as rapidly interconverting conformational ensembles of slightly different functional/nonfunctional forms.…”

Section: Introductionmentioning

confidence: 99%

“…Understanding of allostery has been constantly expanding since seminal studies in the 1960s 18 and 1980s, 19 with the topic being extensively reviewed and reformulated. 4,7-10,20-24…”

Section: Introductionmentioning

confidence: 99%

Decrypting the languages of allostery in membrane-bound K-Ras4B using four complementaryin silicoapproaches

Castelli

Marchetti²,

Serapian

et al. 2023

Preprint

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Proteins are evolved molecular machines whose diverse biochemical functions are (also) dynamically regulated by allostery, through which even faraway protein residues can conformationally communicate. Allostery can express itself in different ways, akin to different "languages": allosteric control pathways predominating in an unperturbed protein are superseded by others as soon as a perturbation arises—e.g, mutation—that alters its function (pathologically or not). Accurately modeling these often-unintuitive phenomena could therefore help explain functional changes in a specific protein whenever they are unclear. Unbiased molecular dynamics (MD) simulations are a possibility; however, since allostery can operate at longer timescales than those accessible by MD, simulations require integration with a reliable method able to, e.g., detect regions of incipient allosteric change, or likely perturbation pathways. Several such (valid) methods exist, but are typically applied singularly, only yielding a partial ("monolinguistic") picture of allostery: we argue their joint application could significantly enrich this picture. To prove this, we perform unbiased MD simulations (~100 μs) of the widely studied, allosterically active oncotarget K-Ras4B, solvated and embedded in a phospholipid membrane, proceeding to decrypt its allostery using four showcase "languages": Distance Fluctuation analysis and the Shortest Path Map capture allosteric communication hotspots at equilibrium; Anisotropic Thermal Diffusion and Dynamic Non-Equilibrium MD assess them once the GTP that oncogenically activates K-Ras4B is, respectively, either superheated or hydrolyzed. "Languages" provide a uniquely articulate, mutually coherent, experimentally consistent picture of allostery in K-Ras4B. At equilibrium, pathways stretch from the membrane-embedded hypervariable region all the way to the active site, touching known flexible allosteric "switches" and proposed pockets. Upon GTP cleavage/perturbation, known to inactivate K-Ras4B, allosteric signals most reverberate on switches and interfaces that recruit effector proteins. Our work highlights the benefits of integrating different allostery detection methods, even in conjunction with unbiased MD.

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Allostery Frustrates the Experimentalist

Cited by 14 publications

References 64 publications

An intramolecular energetic network regulates ligand recognition in a SH2 domain

An intramolecular energetic network regulates ligand recognition in a SH2 domain

Decrypting Allostery in Membrane-Bound K-Ras4B Using Complementary In Silico Approaches Based on Unbiased Molecular Dynamics Simulations

Decrypting the languages of allostery in membrane-bound K-Ras4B using four complementaryin silicoapproaches

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