The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85

Abstract

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

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

“…This result, together with data obtained from equilibrium unfolding experiments, suggests a mechanism in which the intermediate is a high-energy species, never accumulating along the reaction. On the basis of the data of this work and of previous works on the folding of SH2 domains, [33][34][35][36][37] our findings support the hypothesis that the presence of multiple energetic minima in the energy landscape of SH2 domains is not necessary for productive folding and may be only ascribable to an intrinsic propensity selected by the sequence of a single domain to form partially folded state. Future works will clarify the possible general validity of this mechanism.…”

“…Despite their abundance in the proteome, our knowledge about the folding mechanism of SH2 domains is very limited, with only few experimental data available. [33][34][35][36][37][38] Given their key role in cell physiology and their involvement in several human diseases, understanding the determinants of SH2 domain stability, strictly correlated with proper folding and accurate function in the recognition of specific ligands, appears a fundamental task to complete. To achieve this goal, a powerful methodology to determine the biophysical properties of a given protein system relies in its comparison with other proteins belonging to the same family, generally characterized by similar topology and function but with different primary structures.…”

“…Despite their abundance in the proteome, our knowledge about the folding mechanism of SH2 domains is very limited, with only few experimental data available 33–38 . Given their key role in cell physiology and their involvement in several human diseases, understanding the determinants of SH2 domain stability, strictly correlated with proper folding and accurate function in the recognition of specific ligands, appears a fundamental task to complete.…”

Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers

“…32 Like nonproline peptide bonds, there is still a preference for the trans proline isomer. 25 The cis conformer is rare in native proteins yet can be found more frequently in the denatured state, 33 or systems with multiple prolines. 34,35 Earlier TIMS experiments with L-Plrn2 resolved two features in the TIMS mobilogram.…”

SH2 Domains: Folding, Binding and Therapeutical Approaches

“…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.…”