The Abl SH2‐kinase linker naturally adopts a conformation competent for SH3 domain binding

Abstract

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

Abl N-Terminal Cap Stabilization of SH3 Domain Dynamics

“…The width of each isotopic distribution at each deuterium labeling time point in Abl SH3 and Abl NCap3 was measured ( Figure 3B, right). The Abl SH3 alone showed peak broadening characteristic of EX1 unfolding with halflife of 4.61 minutes, as shown previously (12). The unfolding half-life of NCap3 (5.13 min) was very similar to the unfolding half-life of Abl SH3 without the NCap.…”

“…NCap3, NCap32, and NCap32L proteins were overexpressed and purified as described previously (12). Abl core was purified from Sf9 insect cells upon co-expression with YopH, a protein tyrosine phosphatase.…”

“…We previously used HX MS to show that the Abl SH3 domain undergoes partial cooperative unfolding that is sensitive to ligand binding (12). By monitoring the appearance of the unfolding event during the deuterium labeling time-course, the half-life (t 1/2 ) of unfolding can be determined.…”

“…By monitoring the appearance of and changes to the isotopic pattern (which reveals the unfolding event) in various constructs over time, intramolecular interactions involving SH3 can be ascertained. Ligand binding to SH3 substantially slows the unfolding rate and the relative strength of binding can be determined by the magnitude of the slowing (12). Protein dynamics that are altered in general by global stabilization of the structure in an allosteric fashion will also change the deuterium incorporation and peak appearance and can also be monitored.…”

“…In theory, there should be some compensation in c-Abl regulation to account for the loss of SH2 domain interactions, assuming that SFKs and Abl function in similar ways. We have previously shown (12) that the Abl SH2-kinase linker intramolecularly associates with the Abl SH3 domain in the absence of the kinase domain whereas in the SFK Hck, no such interaction could be detected (13). This observation suggests that the c-Abl SH3 domain may have a greater regulatory influence than its SH3 domain counterparts in SFKs.…”