Determining folding and binding properties of the C‐terminal <scp>SH2</scp> domain of <scp>SHP2</scp>

Nardella, Caterina; Malagrinò, Francesca; Pagano, Livia; Rinaldo, Serena; Gianni, Stefano; Toto, Angelo

doi:10.1002/pro.4201

Cited by 7 publications

(28 citation statements)

References 47 publications

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“…Although their fundamental role for the physiology of the cell and their involvement in several molecular pathways, only few experimental works characterized the folding properties of SH2 domains. 10,13,[18][19][20][21] Understanding the determinants of thermodynamic stability as well as characterizing the folding pathway of a protein is of fundamental importance to depict the molecular basis of its biochemical function. Moreover, a structural characterization of the transition state(s) and of possible intermediates along the folding reaction allows to pinpoint potential aberrant misfolding events, that may result in protein misfunction.…”

Section: Discussionmentioning

confidence: 99%

“…In a recent work, by employing a combination of equilibrium and kinetic (un)folding experiments conducted at different experimental conditions, we analyzed the folding mechanism of the C-SH2 domain of SHP2. 13 An analysis of initial and final fluorescence of the unfolding reaction of the wild-type C-SH2 domain as function of [UREA] showed the dependence of the initial fluorescence (which resembles the signal of native protein) to be linear, denoting the absence of burst-phase unfolding events. This aspect allowed us to conclude that the rollover in the unfolding arm of the chevron plot could be ascribable to a folding mechanism implying a change in the rate-limiting step at high denaturant concentrations, with the presence of two transition states along the reaction pathway, and a high-energy intermediate.…”

Section: φ-Value and Linear Free Energy Relationship Analysis Of The ...mentioning

confidence: 99%

“…A powerful strategy to infer the mechanism of folding of a given protein relies in comparing its folding properties with proteins belonging to the same family, that is, sharing an overall same topology but different primary structures 3,11,12 . In a recent work, we demonstrated the C‐SH2 domain to fold through a three‐state mechanism characterized by the presence of a high‐energy intermediate 13 . In this paper, we report a Φ‐value analysis of the C‐terminal SH2 domain of SHP2.…”

Section: Introductionmentioning

confidence: 99%

“…3,11,12 In a recent work, we demonstrated the C-SH2 domain to fold through a three-state mechanism characterized by the presence of a high-energy intermediate. 13 In this paper, we report a Φ-value analysis of the Cterminal SH2 domain of SHP2. By performing an extensive site-directed mutagenesis and a kinetic analysis we were able to characterize the structure of the early and late transition states of the folding reaction.…”

Section: Introductionmentioning

confidence: 99%

“…The dependences of the logarithm of the observed rate constants (k obs ) as function of the concentration of denaturant (chevron plots) of all the variants explored are reported in Figure1. In analogy to our previous work on the C-SH2 domain,13 chevron plots displayed a deviation from linearity in their unfolding arm and were analyzed by using an equation taking into account the presence of a high-energy intermediate along the reaction pathway…”

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Characterization of early and late transition states of the folding pathway of a SH2 domain

et al. 2022

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Albeit SH2 domains are abundant protein-protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C-terminal SH2 domain of SHP2, conducted through a combination of sitedirected mutagenesis and kinetic (un)folding experiments (Φ-value analysis).The energetic profile of the folding reaction of the C-SH2 domain is described by a three-state mechanism characterized by the presence of two transition states and a high-energy intermediate. The production of 29 site-directed variants allowed us to calculate the degree of native-like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ-values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N-terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.

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Section: Discussionmentioning

confidence: 99%

Section: φ-Value and Linear Free Energy Relationship Analysis Of The ...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Characterization of early and late transition states of the folding pathway of a SH2 domain

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Determining folding and binding properties of the C‐terminal SH2 domain of SHP2

et al. 2021

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SH2 domains are a class of protein-protein interaction modules with the function to recognize and bind sequences characterized by the presence of a phosphorylated tyrosine. SHP2 is a protein phosphatase involved in the Ras-ERK1/2 signaling pathway that possess two SH2 domains, namely, N-SH2 and C-SH2, that mediate the interaction of SHP2 with various partners and determine the regulation of its catalytic activity. One of the main interactors of the

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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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Determining folding and binding properties of the C‐terminal SH2 domain of SHP2

Cited by 7 publications

References 47 publications

Characterization of early and late transition states of the folding pathway of a SH2 domain

Characterization of early and late transition states of the folding pathway of a SH2 domain

Determining folding and binding properties of the C‐terminal SH2 domain of SHP2

An intramolecular energetic network regulates ligand recognition in a SH2 domain

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