CBS domains 1 and 2 fulfill different roles in ionic strength-sensing of the ABC transporter OpuA

Akira Karasawa¹, Guus B. Erkens², Ronnie P-A Berntsson³, Renee Otten⁴, Gea K. Schurrman-Wolters⁵, Frans A.A. Mulder⁶, Bert Poolman⁷

1 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands

2 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands

3 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands

4 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands

5 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands

6 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands

7 Departments of Biochemistry and Biophysical Chemistry University of Groningen, The Netherlands, b.poolman@rug.nl

Abstract

Cell volume regulation is an essential function of any cell to overcome the consequences of osmotic stress. Osmoregulatory transporters respond to hyper-osmotic stress by taking up compatible solutes such as glycine betaine, carnitine, trimethylamine N-oxide, or proline, and thereby restore the volume of the cell. For the osmoregulatory ABC transporter OpuA, it has been shown that the regulation involves the sensing of the intracellular ionic strength. In fact, the CBS module of OpuA in conjunction with an anionic membrane surface acts as sensor of internal ionic strength, which allows the protein to respond to osmotic stress. OpuA consists of two different polypeptides: OpuABC with the substrate-binding domain fused C-terminal of the transmembrane domain (TMD), and OpuAA with tandem CBS domains fused C-terminal of the nucleotide-binding domain (NBD). The active OpuA complex consists of two OpuABC and two OpuAA subunits. The structure of the substrate-binding domain has been solved at 1.9Å resolution, and glycine betaine binding to OpuA has been shown to be independent of ionic strength. The tandem CBS domains of the OpuAA subunit each have predicted β-α-β-β-α secondary structure and crystal structures of analogous proteins suggest a dimeric state, consistent with the dimeric structure of OpuA. We have now engineered the CBS module of OpuA and probed the effects of domain modifications and cross-linking on the ionic regulation of transport. Each of the modified proteins was purified and reconstituted into liposomes and bilayer nanodiscs of defined lipid composition, and the transport and ATPase activity was determined as a function of the ionic strength and other parameters. We show that CBS2-CBS2 interface residues are critical for transport activity and/or ionic regulation of transport, whereas CBS1 serves no functional role. We establish that Cys residues in CBS1, CBS2, and NBD are more accessible for cross-linking at high than low ionic strength, indicating that these domains undergo conformational changes when transiting between the active and inactive state. Structural analyses (NMR, CD and light-scaterring measurements) suggest that the CBS module is largely unstructured. Moreover, we could substitute CBS1 by a linker and preserve ionic regulation of transport. The data suggest that CBS1 serves as linker and the structured CBS2-CBS2 interface forms a hinge point for ionic strength-dependent rearrangements that are transmitted to the NBD and thereby affect translocation activity.

DOI^®: 10.3288/contoo.paper.1718