Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.

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 ?4 (T?4) 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 (1)H-(15)N HSQC spectra of T?4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of T?4 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/T?4 complex that shows great accordance with the reference structure.

SUBMITTER: Huang S 

PROVIDER: S-EPMC5031973 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature