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

Abstract

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.

Folding and Binding Kinetics of the Tandem of SH2 Domains from SHP2

“…Here, we aim to provide such information by employing folding and binding kinetic experiments of the NSH2-CSH2 tandem domains explored through fast kinetic experiments. Building upon previously obtained results on the isolated domains [ 33 , 34 , 35 , 36 ], we provide a comprehensive characterization of the folding mechanism of the NSH2-CSH2 tandem explored at different pH conditions. The analysis of data highlights that, while in most of the conditions explored, the two domains appear to fold and unfold independently one to the other, more complex scenarios arise at acidic pH.…”

From covalent transition states in chemistry to noncovalent in biology: from β- to Φ-value analysis of protein folding

“…I have indicated (nc) for some that appear to fold by nucleation condensation and (fw) by framework. Monomeric λ-repressor (Burton et al, 1998), ADA2H (nc) (Villegas et al, 1998; Kukic et al, 2017), acyl-coA binding protein (nc) (Kragelund et al, 1999), SH3 domains (α-spectrin (nc), src) (Grantcharova et al, 1998; Martinez and Serrano, 1999; Guerois and Serrano, 2000), SH3 domain from Grb2 (nc) (Troilo et al, 2018), acylphosphatase (nc) (Chiti et al, 1999), Im7 and Im9 (Friel et al, 2003; Paci et al, 2004; Bartlett and Radford, 2010), NTL9 domain of L9 (nc) (Anil et al, 2005; Sato et al, 2017), cheY (1 domain nc) (Lopez-Hernandez and Serrano, 1996), Sod1 (Yang et al, 2018), S6 (Otzen and Oliveberg, 2002; Lindberg et al, 2006), U1a (nc) (Ternstrom et al, 1999), azurin (Wilson and Wittung-Stafshede, 2005; Zong et al, 2006), apo-flavodoxin (Campos et al, 2004; Muralidhara et al, 2005; Bueno et al, 2006; Lopez-Llano et al, 2006; Homouz et al, 2009; Stagg et al, 2010; Galano-Frutos et al, 2022), WW domains (polarised) (Jager et al, 2001; Petrovich et al, 2006; Dave et al, 2016), villin headpiece (Cho et al, 2010), SH2 domains (Visconti et al, 2019; Toto et al, 2022), HYPA/FBP11 FF domain (bn) (Jemth et al, 2005), PTP-BL, PDZ2 and PSD-95 PDZ3 (3-state) (Calosci et al, 2008), ubiquitin (nc) (Went and Jackson, 2005; Varnai et al, 2008), RNaseA (nc) (Font et al, 2006), yACBP and 3 variants of bACBP (Teilum et al, 2005), B domain of Protein A (nc) (Sato et al, 2006), α -lactalbumin (Chedad et al, 2005), R15 (nc) R16 (fw) R17 (fw) (domains of chicken brain α -spectrin) (Wensley et al, 2009), TI I27 (Fowler and Clarke, 2001), TNfn3 (Geierhaas et al, 2004), LysM domain (Nickson et al, 2008), FKB12 (nc) (Main et al, 2001), apocyotchrome b562 (Zhou et al, 2005), SAP (Dodson and Arbely, 2015), knotted proteins (Mallam et al, 2008; Jackson et al, 2017), raf (polarised) (Campbell-Valois and Michnick, 2007), four-helix HYPA/FPB11(Jemth et al, 2005), tumour suppressor P16 (Tang et al, 2003), barstar (nc) (Nolting et al, …”

“…S6 (Otzen and Oliveberg, 2002;Lindberg et al, 2006), U1a (nc) (Ternstrom et al, 1999), azurin (Wilson andZong et al, 2006), apo-flavodoxin (Campos et al, 2004;Muralidhara et al, 2005;Bueno et al, 2006;Lopez-Llano et al, 2006;Homouz et al, 2009;Stagg et al, 2010;Galano-Frutos et al, 2022), WW domains (polarised) (Jager et al, 2001;Petrovich et al, 2006;Dave et al, 2016), villin headpiece (Cho et al, 2010), SH2 domains (Visconti et al, 2019;Toto et al, 2022), HYPA/FBP11 FF domain (bn) (Jemth et al, 2005), PTP-BL, PDZ2 and PSD-95 PDZ3 (3-state) (Calosci et al, 2008), ubiquitin (nc) (Went and Jackson, 2005;Varnai et al, 2008), RNaseA (nc) (Font et al, 2006), yACBP and 3 variants of bACBP (Teilum et al, 2005), B domain of Protein A (nc) (Sato et al, 2006), α-lactalbumin (Chedad et al, 2005), R15 (nc) R16 (fw) R17 (fw) (domains of chicken brain α-spectrin) (Wensley et al, 2009), TI I27 (Fowler and Clarke, 2001), TNfn3 (Geierhaas et al, 2004), LysM domain (Nickson et al, 2008), FKB12 (nc) (Main et al, 2001), apocyotchrome b562 (Zhou et al, 2005), SAP (Dodson and Arbely, 2015), knotted proteins (Mallam et al, 2008;Jackson et al, 2017), raf (polarised) (Campbell-Valois and Michnick, 2007), four-helix HYPA/FPB11 …”

SH2 Domains: Folding, Binding and Therapeutical Approaches

“…A powerful methodology that can be applied to obtain such information is the φ-value analysis, an approach that relies on extensively mutating individual residues of the domain and monitoring the effect of mutations on the folding kinetics, inferring the role of each residue in the folding reaction and normalizing it in terms of native-like interactions in the probed state (details about the φ-value analysis methodology can be found at [17,18]). Both N-SH2 and C-SH2 were subjected to a φ-value analysis [19,20] (Figure 1). The analysis of φ-values obtained for the N-SH2 domain revealed that the intermediate state (early event of folding) is highly structured, with native-like interactions further locked in place in the late transition state.…”

“…and normalizing it in terms of native-like interactions in the probed state (details about the ϕ-value analysis methodology can be found at [17,18]). Both N-SH2 and C-SH2 were subjected to a ϕ-value analysis [19,20] (Figure 1). The analysis of ϕ-values obtained for the N-SH2 domain revealed that the intermediate state (early event of folding) is highly structured, with native-like interactions further locked in place in the late transition state.…”