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Ruggiero MJ , Malhotra S , Fenton AW , Swint-Kruse L , Karanicolas J , Hagenbuch B
A clinically-relevant polymorphism in the Na(+)/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position
J Biol Chem. 2021 Jan-June;296 :100047
PMID: 33168628    PMCID: PMC7948949    URL: https://www.ncbi.nlm.nih.gov/pubmed/33168628
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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 non-conserved 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 cotransporting polypeptide (NTCP), at the site of a pharmacologically-relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions ("S267X") with three substrates (taurocholate, 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 suite to model structure and stability changes of S267X. Although buried near the substrate binding site, S267X substitutions were easily accommodated in the NTCP 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.
1083-351x Ruggiero, Melissa J Malhotra, Shipra Fenton, Aron W Swint-Kruse, Liskin Orcid: 0000-0002-5925-9741 Karanicolas, John Orcid: 0000-0003-0300-726x Hagenbuch, Bruno Orcid: 0000-0002-2938-8630 Journal Article United States J Biol Chem. 2020 Nov 9:jbc.RA120.014889. doi: 10.1074/jbc.RA120.014889.