Functional characterization of the Υ-aminobutyric acid transporter from mycobacterium smegmatis MC² 155 reveals sodium-driven GABA transport

TY - JOUR

T1 - Functional characterization of the Υ-aminobutyric acid transporter from mycobacterium smegmatis MC2 155 reveals sodium-driven GABA transport

AU - Pavić, Ana

AU - Ji, Yurui

AU - Serafini, Agnese

AU - Garza-Garcia, Acely

AU - McPhillie, Martin J.

AU - Holmes, Alexandra O. M.

AU - Sorio de Carvalho, Luiz Pedro

AU - Wang, Yingying

AU - Bartlam, Mark

AU - Goldman, Adrian

AU - Postis, Vincent L. G.

N1 - Funding Information: We are grateful for financial support from the Leeds Beckett University (to A.P. and V. L.G.P.), the China Scholarship Council (to Y.J.), the Wellcome Trust (ref.019322/7/10/Z), the Erkko Foundation, and the BBSRC (BB/M021610/1) (to A.G.). Work in the Mycobacterial Metabolism and Antibiotic Research Laboratory was chiefly supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (grant no. FC0010060), the UKRI Medical Research Council (grant no. FC0010060), and the Wellcome Trust (grant no. FC0010060), and also by Wellcome Trust New Investigator Award 104785/B/14/Z (to L.P.S.D.C.). We also thank the National Science Publisher Copyright: © 2021 Pavi_c et al.

PY - 2021/1/25

Y1 - 2021/1/25

N2 - Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential g-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SECMALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported g-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of b-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that g-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that g-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active g-aminobutyric acid transport protein from mycobacteria.

AB - Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential g-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SECMALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported g-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of b-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that g-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that g-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active g-aminobutyric acid transport protein from mycobacteria.

KW - GABA

KW - Membrane biology

KW - Mycobacteria

KW - Transporter

UR - http://europepmc.org/abstract/med/33288625

UR - https://www.scopus.com/pages/publications/85100445505

U2 - 10.1128/JB.00642-20

DO - 10.1128/JB.00642-20

M3 - Article

C2 - 33288625

SN - 0021-9193

VL - 203

JO - Journal of Bacteriology

JF - Journal of Bacteriology

IS - 4

M1 - e00642-20

ER -