Mapping Motions and Structure to a State Necessary for Oligomerization of a Large GTPase: A Joint SAXS, NSE, EPR and FRET Study

Abstract

By combining double electron-electron resonance (EPR), small angle x-ray scattering (SAXS) and time-resolved fluorescence-measurements (TCSPC) we resolved two distinct conformational states in hGBP1 a large GTPase composed out of three domains, a GTPase-, a middle- and a helical-domain. We find that the C-terminal helix of the helical domain, which is important for oligomerization, has two distinct binding modes on the GTPase domain. By neutron spin echo spectroscopy we show that the correlation time of the conformational change is slower than nanoseconds. By FRET lifetime filtered species cross-correlation (fFCS) we fully characterize the relaxation time distribution of the conformational changes and find that it happens mainly in the microsecond-regime. By multiple FRET-fFCS measurements we determined the internal flexibility of the protein and find that the C-terminal helices a12/a13 are highly flexible relatively to the middle domain. This finding is consistent with the two conformational changes that involve a domain-flip of the middle-domain and the C-terminal helices relatively to the LG-domain and in line with molecular dynamics simulations. We can describe most observations by a two state system. However, the amplitude decay of the relaxation time spectrum suggests that the conformational change happens on a rugged energy landscape. We previously showed that the GTPase- and the C-terminal helices of two hGBP1s associate in presence of GDP-AlFx. If the protein remains in its major state at room temperature an association of the helices a13 is sterically impossible. Hence, we conclude that the minor state at room temperature or a transient state populated during the domain flip is relevant for association of the C-terminal helices a13.