A clinically relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position

Melissa J Ruggiero,Shipra Malhotra,Aron W Fenton,Liskin Swint-Kruse,John Karanicolas,Bruno Hagenbuch

doi:10.1074/jbc.ra120.014889

Melissa J Ruggiero, Shipra Malhotra + Show 4 more

Open Access

https://doi.org/10.1074/jbc.ra120.014889

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Abstract

Conventionally, most amino acid substitutions at “important” protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of nonconserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary “rheostats”. Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate (TCA) cotransporting polypeptide, at the site of a pharmacologically relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (S267X) with three substrates (TCA, estrone-3-sulfate, and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax) and substrate specificity. To assess protein stability, we measured surface expression and used the Rosetta software (https://www.rosettacommons.org) suite to model structure and stability changes of S267X. Although buried near the substrate-binding site, S267X substitutions were easily accommodated in the Na+/TCA cotransporting polypeptide structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.

Highlights

Amino acid substitutions are commonly used to evaluate which amino acids in a protein contribute to function
If rheostat positions do exist in integral membrane proteins, we wondered whether the functional outcomes arising from various substitutions were dependent on the substrate being transported
Because generalizable features for identifying rheostat positions have not yet been validated, we first faced the challenge of identifying a likely candidate among the 349 amino acid positions of Na+/taurocholate (TCA) cotransporting polypeptide (NTCP)

Summary

Introduction

Amino acid substitutions are commonly used to evaluate which amino acids in a protein contribute to function. If rheostat positions do exist in integral membrane proteins, we wondered whether the functional outcomes arising from various substitutions were dependent on the substrate being transported. The present study provided opportunity to relate the continuum of functional outcomes of the rheostat variants to the complex conformational changes experienced by an integral membrane protein during transport.

Results

Conclusion