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

Abstract

<jats:title>Abstract</jats:title><jats:p>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<jats:sup>+</jats:sup>/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 (K<jats:sub>m</jats:sub> and V<jats:sub>max</jats:sub>). However, the outcomes were substrate dependent, indicating altered 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.</jats:p>

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population

“…The structural data for I98X variants are consistent with the idea that the structure around a rheostat position accommodates a range of side chain types. Similar accommodations were computationally predicted for multiple variants at a rheostat position in the E. coli lactose repressor protein 14 and the sodium taurocholate co‐transporting polypeptide 5 …”

“…Understanding how each position of a protein contributes to function is central to such diverse applications as interpreting exome sequencing in personalized medicine and engineering novel protein functions for biotechnology. We previously showed that many proteins contain a class of amino acid positions with special abilities to alter protein function 1–6 . At these “rheostat” positions, substituting a range of amino acid types results in a striking continuum of functional outcomes.…”

“…Indeed, rheostat positions can be likened to a volume dial on a stereo, with substitutions dialing the functional “volume” either up or down. Furthermore, substitutions at some rheostat positions modulated only a single functional parameter (e.g., K d for binding ligand) whereas substitutions at others simultaneously modulated multiple parameters (e.g., substrate affinity, K d for binding allosteric ligand, and the magnitude of allosteric coupling between them) 1,3–7 …”

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation

“…One of their defining features is that, when individually substituted with a range of amino acids, various functional parameters (e.g., binding affinity, allosteric regulation) are modulated. Indeed, the functional range for each position can span several orders of magnitude 3–7 . As such, substitutions at these “rheostat” positions provide ready opportunities for fine‐tuning function during evolution and protein engineering.…”

“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein

“…As an alternative approach, we agnostically, but systematically, substituted nonconserved positions with a wide variety of amino acids. This strategy revealed a class of functionally-important positions with substitution outcomes that differed strikingly from textbook expectations ( Supplementary Figure 1; (14)(15)(16)(17)(18)(19)(20)). For this new class of "rheostat" positions, a key feature is their ability to modulate function (e.g.…”