8VXU

Crystal structure of Gdx-Clo A60T from Small Multidrug Resistance family of transporters in complex with cetyltrimetylammonium

Summary for 8VXU

• Entry DOI: 10.2210/pdb8vxu/pdb
• Descriptor: Multidrug resistance protein, SMR family, L10 Monobody, CETYL-TRIMETHYL-AMMONIUM (3 entities in total)
• Functional Keywords: small multidrug resistance, smr, quaternary ammonium, transport protein
• Biological source: Clostridia bacterium; More
• Total number of polymer chains: 8
• Total formula weight: 87618.81

Authors

Burata, O.E.,Stockbridge, R.B. (deposition date: 2024-02-06, release date: 2024-05-08, Last modification date: 2024-06-26)

Primary citation

Burata, O.E.,O'Donnell, E.,Hyun, J.,Lucero, R.M.,Thomas, J.E.,Gibbs, E.M.,Reacher, I.,Carney, N.A.,Stockbridge, R.B.
Peripheral positions encode transport specificity in the small multidrug resistance exporters.
Proc.Natl.Acad.Sci.USA, 121:e2403273121-e2403273121, 2024

PubMed Abstract: In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

PubMed: 38865266
DOI: 10.1073/pnas.2403273121

Experimental method

X-RAY DIFFRACTION (2.29 Å)