Differential modulation of collybistin conformational dynamics by the closely related GTPases Cdc42 and TC10

Abstract

Interneuronal synaptic transmission relies on the proper spatial organization of presynaptic neurotransmitter release and its reception on the postsynaptic side by cognate neurotransmitter receptors. Neurotransmitter receptors are incorporated into and arranged within the plasma membrane with the assistance of scaffolding and adaptor proteins. At inhibitory GABAergic postsynapses, collybistin, a neuronal adaptor protein, recruits the scaffolding protein gephyrin and interacts with various neuronal factors including cell adhesion proteins of the neuroligin family, the GABAA receptor α2-subunit and the closely related small GTPases Cdc42 and TC10 (RhoQ). Most collybistin splice variants harbor an N-terminal SH3 domain and exist in an autoinhibited/closed state. Cdc42 and TC10, despite sharing 67.4% amino acid sequence identity, interact differently with collybistin. Here, we delineate the molecular basis of the collybistin conformational activation induced by TC10 with the aid of recently developed collybistin FRET sensors. Time-resolved fluorescence-based FRET measurements reveal that TC10 binds to closed/inactive collybistin leading to relief of its autoinhibition, contrary to Cdc42, which only interacts with collybistin when forced into an open state by the introduction of mutations destabilizing the closed state of collybistin. Taken together, our data describe a TC10-driven signaling mechanism in which collybistin switches from its autoinhibited closed state to an open/active state.

Deciphering the conformational dynamics of gephyrin-mediated collybistin activation

“…Previous studies demonstrated that activation of CB upon interacting with NL2 icd or TC10 leads to an open structure of CB, which allows CB to interact with phosphoinositides located in the postsynaptic membrane ( 29 , 33 , 34 , 55 ). The two-state Gaussian distributed distance fit of the CB F1 DA sensor showed an increase in the population of the low-FRET state upon interaction with NL2 icd , indicating NL2 icd -mediated CB opening.…”

“…In contrast, the SH3 domain lacking CB2 splice variant (CB2-SH3 − ) constitutively regenerates the GTP-bound state of Cdc42 ( 30 , 31 , 32 ). However, a recent study demonstrated that only TC10, another Dbl family GEF that is closely related to Cdc42 but not Cdc42, can effectively mediate the conformational activation of full-length CB ( 33 ). Interestingly, earlier biochemical and cellular studies ( 29 , 34 ) indicated that CB interaction with the cytosolic region of NL2 led to a similar activation of CB ( Fig.…”