Deciphering the conformational dynamics of gephyrin-mediated collybistin activation

Abstract

Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery and at inhibitory GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl-family guanine nucleotide exchange factor and neuronal adaptor protein. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin FRET sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction, which does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal a dynamic behavior of the SH3 domain already in the closed state of collybistin. Finally, our data provide strong evidence for a collybistin-gephyrin communication network, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.

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

“…In order to examine the role of the two GTPases in CB activation, we employed previously described fluorescence lifetime-based CB FRET sensors ( Imam et al, 2022 ) derived from the CB2-SH3 + splice variant ( Supplementary Figure 1A ). We recombinantly purified TC10 and Cdc42 ( Supplementary Figures 1B,C ) and incubated both proteins in a 100-fold molar excess (100 μM) with the CB wild-type FRET sensors ( Imam et al, 2022 ).…”

“…In order to examine the role of the two GTPases in CB activation, we employed previously described fluorescence lifetime-based CB FRET sensors ( Imam et al, 2022 ) derived from the CB2-SH3 + splice variant ( Supplementary Figure 1A ). We recombinantly purified TC10 and Cdc42 ( Supplementary Figures 1B,C ) and incubated both proteins in a 100-fold molar excess (100 μM) with the CB wild-type FRET sensors ( Imam et al, 2022 ). For interaction studies, we individually measured the average fluorescence lifetime (⟨τ⟩) of CFP ( Figure 2A ) of the CB FRET sensor (F1 D0 ) and its FlAsH-labeled counterpart (F1 DA ), in the absence and presence of either TC10 or Cdc42.…”

“…The C-terminal TC10 deletion variant, TC10ΔC, was constructed by deleting the last 20 amino acids by using RF cloning. The full-length Cdc42 construct has been previously described ( Xiang et al, 2006 ) as have the wild-type CB FRET sensor (F1 D0 ), open state mutant sensors (F1 smD0 and F1 dmD0 ) and the series of additional CB FRET sensors ( Imam et al, 2022 ). All FRET sensors are derived from the CB2-SH3 + variant.…”

“…Eluted protein fractions were concentrated to 10–12 mg/ml by ultrafiltration, flash frozen and stored at −80°C for later usage. All CB FRET sensors were purified as described ( Imam et al, 2022 ) as was full-length Cdc42 ( Xiang et al, 2006 ), in this case with minor modifications. All CB FRET sensors were FlAsH labeled as described ( Imam et al, 2022 ).…”

“…All CB FRET sensors were purified as described ( Imam et al, 2022 ) as was full-length Cdc42 ( Xiang et al, 2006 ), in this case with minor modifications. All CB FRET sensors were FlAsH labeled as described ( Imam et al, 2022 ).…”