Abstract
Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure.
Highlights
The NMR method has been utilized to investigate the interactions between G-actin and actin binding proteins (ABPs) with both strong and weak binding affinities, and provided information concerning the conformational changes of ABPs upon binding to G-actin, and the G-actin binding sites on ABPs9–12
A recent study succeeded in the NMR observation of the sidechain methyl group of G-actin expressed in Pichia pastoris[16], it is impossible to determine the accurate 3D structure of the G-actin/ABP complex, due to lack of intermolecular distance information between the ABP and G-actin
To perform the paramagnetic relaxation enhancement (PRE) experiments, G-actin mutants with a single reactive cysteine residue were prepared by the baculovirus expression system
Summary
The NMR method has been utilized to investigate the interactions between G-actin and ABPs with both strong and weak binding affinities, and provided information concerning the conformational changes of ABPs upon binding to G-actin, and the G-actin binding sites on ABPs9–12. Based on previous NMR and X-ray crystallographic studies, it was demonstrated that Tβ4 adopts an elongated conformation in the G-actin bound state, and forms two amphipathic helices at the N- and C-termini[10,12,23]. These N- and C-terminal helices bind to the barbed end and the pointed end of G-actin, respectively, thereby inhibiting the G-actin monomer from polymerizing into existing actin-filaments. Using the spin labeled actin, we performed PRE experiments and collected intermolecular distance information between G-actin and ABP, based on the line-broadening of the NMR signals of ABP. We computed the docking structures of the actin/ABP complex, using the intermolecular distance information as distance constraints
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
