title: 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 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 the1H-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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shuxian
authors.initials: S
authors.lastname: Huang
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Umemoto
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tamura
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kofuku
authors.affiliation: Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Taro Q P
authors.initials: TQ
authors.lastname: Uyeda
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Nishida
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.|Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada