Folding and Binding Mechanisms of the SH2 Domain from Crkl

Abstract

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains.

An intramolecular energetic network regulates ligand recognition in a SH2 domain

“…On the other hand, a greater contribution of the microscopic dissociation rate constant on the stability of the complex is evident, with k off increasing at higher K D values (the slope of log k off vs. log K D being 0.76 ± 0.08, with a R 2 = 0.75). Given the nature of designed site‐directed variations, occurring only on hydrophobic side‐chains and without perturbing polar and charged residues (Fersht & Sato, 2004 ), this result well correlates with a scenario in which the early recognition events are driven by electrostatic contributions, while non‐polar interactions lock the complex in place in the late events of binding, in analogy to what has been previously described for other SH2 domains (Bonetti et al, 2018 ; Nardella et al, 2021 ; Nardella et al, 2022 ; Visconti, Malagrinò, et al, 2020 ; Visconti, Toto, et al, 2020 ).…”

SH2 Domains: Folding, Binding and Therapeutical Approaches

“…In fact, most conserved residues are clustered on the βB strand, where a conserved arginine residue in the FLVR motif (Figure 2B) (Arg βB5 or Arg175 in the v-Src SH2 domain) plays the central role in forming a double hydrogen bond with the phosphate group of pTyr [45,46]. The binding affinities of the SH2 domain for the phosphotyrosine peptide are reported to be in the range of what is typically observed for protein-protein interactions [26], ranging from 10 −5 to 10 −8 M [16,24,25,47]. Additional residues that are key for phosphopeptide binding are His βD4, Lys βD6, and Arg αA2, which coordinate and anchor the aromatic ring of the phospho-tyrosine [48].…”

“…However, quantitative analysis of kinetic data highlights a generally conserved folding mechanism between the C-SH2 and N-SH2 domains. Other SH2 domains that have been characterized in their folding properties are the SH2 domain of Src [21], the N-terminal and C-terminal SH2 domains of the p85 subunit of PI3K [22,23], and the SH2 domain of Crkl [24]. It is interesting that the SH2 domain from Crkl displays similar folding kinetics compared to the N-SH2 domain of SHP2, with a pronounced roll-over effect in the refolding arm of the chevron plot being compatible with an energetic profile implying the presence of an obligatory intermediate accumulating along the reaction.…”

Per Aspera ad Chaos: Vladimir Uversky’s Odyssey through the Strange World of Intrinsically Disordered Proteins

“…Understanding the subtle mechanisms by which such interactions are triggered and regulated is of paramount importance to decipher their pathophysiological role, but also for exploring the potential for pharmacological approaches targeting IDPs. Under this theme, readers will find five scientific articles and one review [ 17 , 18 , 19 , 20 , 21 , 22 ].…”

“…Understanding the subtle mechanisms by which such interactions are triggered and regulated is of paramount importance to decipher their pathophysiological role, but also for exploring the potential for pharmacological approaches targeting IDPs. Under this theme, readers will find five scientific articles and one review [17][18][19][20][21][22]. Predicting and modeling of IDPs by conventional and advanced bioinformatic tools-Significant progress has been made over the past decade in the development of bioinformatics tools for predicting and modeling structural disorder.…”