Many autoinhibited signaling proteins are responsive to small changes in the populations of cognate binding partners1–5
. The dynamic equilibrium between autoinhibited and activated-like conformations of signaling proteins in the absence of activators is believed to play a key role in achieving the seemingly contradictory tasks of preventing uncontrolled activation and enabling prompt response to cellular stimuli6,7
. This model implies that the interdomain interface responsible for autoinhibition must be sufficiently stable to prevent constitutive activation, but sufficiently labile to allow facile kinetic exchange between autoinhibited and activation-competent states.
The conformation of a protein in complex with a target molecule must, in the absence of the target, have a free energy equal to or greater than that of the experimentally observed apo structure. Consequently, the binding affinity for the target must be sufficient to overcome the energetic penalty associated with forming higher energy holo conformations8
. The thermodynamics and kinetics of such conformational changes are critical for understanding the physical principles controlling binding affinity; however, detection of low populations of high energy states in the absence of binding partners represents a considerable experimental challenge.
A full description of the molecular mechanisms responsible for controlling the dynamic equilibrium between autoinhibited (closed) and activated-like (open) states requires comparison of the structures and thermodynamics of individual domains in isolation and in the context of full-length molecules, as well as knowledge of the kinetics of interconversion between the various populated conformational states. Moreover, unique spectroscopic probes are needed for studying the individual domains within the full-length multi-domain protein9,10
. Consequently, complete descriptions are not yet available of the molecular basis for control of autoinhibition of multi-domain signaling proteins.
Archetypical signaling adaptor proteins of the Crk family are comprised of Src homology 2 (SH2) and 3 (SH3) domains and have been implicated in many cellular processes including cell motility, proliferation and adhesion11–14
(). Crk proteins mediate diverse protein-protein interactions, through their SH2 and SH3 domains, in downstream transduction of growth and differentiation signals11
. Crk-II consists of an N-terminal SH2 domain and two SH3 domains (SH2-nSH3-cSH3). The binding of the N-terminal SH3 (nSH3) domain to C3G, a guanine-nucleotide exchange factor for small molecular weight GTPase15
contributes to the integrin signaling18
and the regulation of fibroblast migration11
. The biological activity of the nSH3 domain of Crk-II is negatively regulated by the C-terminal SH3 (cSH3) domain via
intramolecular domain-domain interactions11,19
. The solution structure of Crk-II shows that the cSH3 domain stabilizes a closed autoinhibited state of Crk-II in which the ligand binding site of the nSH3 domain is blocked by interdomain interactions with the SH2 domain; however, the active site of the nSH3 domain is not directly occluded by the cSH3 domain19
(). Alternative splicing of the c-crk
gene yields Crk-I (SH2-nSH3), which lacks the C-terminal SH3 domain and is constitutively active, causing transformation of hematopoietic and fibroblast cells12,20
. The NMR solution structure of Crk-I indicates that the active site of the nSH3 domain is exposed to solvent19
, consistent with the inability of the SH2 domain alone to exert autoinhibitory control of nSH3 activity9
. Various models have been proposed for control of autoinhibition of Crk-II19,21,22
; however, a complete biophysical analysis of this process, which necessarily involves analysis of the full-length protein and individual domains, has not been reported. Herein, we present a thermodynamic, kinetic and structural analysis that reveals a critical role for selective domain destabilization in tuning the responsiveness of Crk-II to activation.
Scheme for the autoinhibition and activation of Crk-II