Assembly of the Non-Canonical Myo9a-RhoGAP and RhoA·GDP Transition State Complex in the Presence of MgF3

Fengshuang Yi; Xin Zhang; Ruobing Liang

doi:10.1007/s10930-021-10027-4

Assembly of the Non-Canonical Myo9a-RhoGAP and RhoA·GDP Transition State Complex in the Presence of MgF₃

Protein J. 2021 Dec;40(6):842-848. doi: 10.1007/s10930-021-10027-4. Epub 2021 Oct 28.

Authors

Fengshuang Yi¹, Xin Zhang^{2

3}, Ruobing Liang⁴

Affiliations

¹ Department of Respiratory and Critical Care Medicine, Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China. yifengshuang@ccmu.edu.cn.
² Department of Respiratory and Critical Care Medicine, Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China.
³ Department of Geriatric Respiratory Disease, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China.
⁴ Biologics Discovery, WuXi Biologics, Shanghai, China.

PMID: 34709522
DOI: 10.1007/s10930-021-10027-4

Abstract

Myo9a is an actin-based molecular motor with a RhoGAP domain in its C-terminal tail. It plays a role in a variety of biological processes, such as in regulating the immune response, neuron development, and cancer progression, and its deregulation can lead to the development of disease conditions. Myo9a acts mainly via its RhoGAP domain. In the current study, we used a pET32a vector with an N-terminal Trx-His6 tag to express Myo9a-RhoGAP in a soluble form. High-purity Myo9a-RhoGAP protein was obtained after two rounds of Ni²⁺ affinity and size-exclusion chromatography. We mixed Myo9a-RhoGAP and RhoA in equimolar ratios in the presence of 5 mM MgCl₂ and 20 mM NaF to achieve a stable RhoA GTP hydrolysis transition state complex. Analytical gel filtration and SDS-PAGE were used to verify complex formation. ITC and GAP assays suggested that Myo9a-RhoGAP could bind to RhoA and accelerate RhoA GTP hydrolysis in vitro. We purified the soluble Myo9a-RhoGAP protein with GAP activity and achieved the Myo9a-RhoGAP/RhoA·GDP/MgF₃^- complex assembly in vitro for the first time. The data may provide novel insights into Myo9a structure and function.

Keywords: Assembly in vitro; GAP assay; Magnesium fluoride; Myo9a; Myo9a-RhoGAP; Transition state.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actins*
GTPase-Activating Proteins*

Substances

Actins
GTPase-Activating Proteins
rho GTPase-activating protein