X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Lan Guan,Parameswaran Hariharan

doi:10.1038/s42003-021-02462-x

Abstract

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

Highlights

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals
The glycoside–pentoside–hexuronide:cation symporter family (GPH)[1], which is a subgroup of the major facilitator superfamily (MFS) of membrane transporters found from bacteria to mammals that catalyze a coupled substrate transport with monovalent Na+, Li+, or H+, Na+ or Li+, H+ or Li+, or only H+
Melibiose transport and ligand binding. [3H]Melibiose transport assays with intact E. coli cells confirmed that the D59C MelB of Salmonella typhimurium (MelBSt) mutant lost all three modes of melibiose active transport coupled to H+, Na+, or Li+, even with increased concentration of Na+ or Li+ (Fig. 1a)

Summary

Introduction

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The conserved cation-binding pocket is proposed, which directly connects to the sugar specificity pocket These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A. While active transporters can move specific solutes into cells effectively, a majority of available drugs enter cells by inefficient solubility diffusion This is largely due to lack of knowledge on active transporters, in particular the detailed understanding about substrate-binding sites and transport mechanisms. The glycoside–pentoside–hexuronide:cation symporter family (GPH)[1], which is a subgroup of the major facilitator superfamily (MFS) of membrane transporters found from bacteria to mammals that catalyze a coupled substrate transport with monovalent Na+, Li+, or H+, Na+ or Li+, H+ or Li+, or only H+ Lack of highresolution substrate-bound 3-D structure is a bottleneck for unraveling molecular recognition and transport mechanisms, which significantly hampered the developments towards the pharmaceutical applications as drug targets or as vehicles for drug delivery

Methods

Results

Conclusion