Structural Plasticity Is a Feature of Rheostat Positions in the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP)

Abstract

In the Na⁺/taurocholate cotransporting polypeptide (NTCP), the clinically relevant S267F polymorphism occurs at a "rheostat position". That is, amino acid substitutions at this position ("S267X") lead to a wide range of functional outcomes. This result was particularly striking because molecular models predicted the S267X side chains are buried, and thus, usually expected to be less tolerant of substitutions. To assess whether structural tolerance to buried substitutions is widespread in NTCP, here we used Rosetta to model all 19 potential substitutions at another 13 buried positions. Again, only subtle changes in the calculated stabilities and structures were predicted. Calculations were experimentally validated for 19 variants at codon 271 ("N271X"). Results showed near wildtype expression and rheostatic modulation of substrate transport, implicating N271 as a rheostat position. Notably, each N271X substitution showed a similar effect on the transport of three different substrates and thus did not alter substrate specificity. This differs from S267X, which altered both transport kinetics and specificity. As both transport and specificity may change during protein evolution, the recognition of such rheostat positions may be important for evolutionary studies. We further propose that the presence of rheostat positions is facilitated by local plasticity within the protein structure. Finally, we note that identifying rheostat positions may advance efforts to predict new biomedically relevant missense variants in NTCP and other membrane transport proteins.

Keywords: protein plasticity; rheostat; transmembrane protein.

Figure 1

Predicted stability differences associated with sequence variation at the N271 position. (A) Rosetta energies for wildtype and 18 sequence variants at position 271 of the inward-open model. Proline is not shown, because incorporation of proline into helical segments cannot be reliably modeled with Rosetta. (B) Structural details from the models underlying these energy differences. Four different sequence variants are compared with the wild-type N271; the conformations of TM helices 2, 7, and 10 respond to changes in the amino acid at position 271, which is located on TM helix 9b. (C) Rosetta energies using the outward-open model. Proline is again not included in this analysis. (D) Structural details from the outward-open models. In this conformation, the positions of TM helices 7 and 10 respond to changes in the amino acid at position 271.

Figure 2

Surface expression and quantification of wildtype NTCP and N271X variants. (A) Surface expression of wildtype NTCP and N271X variants on a representative Western blot. Proteins from HEK293 cells transiently transfected with empty vector (EV), wildtype NTCP (WT), and N271 variants were separated using a 4–20% polyacrylamide gradient gel and then transferred to nitrocellulose membranes. Blots were probed simultaneously with Na⁺/K⁺-ATPase as a loading control (100 kDa) and Tetra-His antibodies which detects His-tagged NTCP variants. (B) Quantification of Western blots with N271 variants compared to wildtype NTCP. Three independent surface expression experiments were quantified using Image Studio Lite. Individual data points are shown with the bar representing the mean ± SD. Horizontal lines indicate the upper and lower limit of the wildtype value plus (upper line) and minus (lower line) the standard deviation of the wildtype value. Asterisks denote significant difference from wildtype NTCP with a p < 0.05 level.

Figure 3

Substrate transport by position 271 variants corrected for surface expression. Initial uptake results from Figure S2 were normalized for the surface quantification in Figure 2. Corrected transport is shown with the N271X variants and wildtype NTCP ordered from greatest to least uptake for each substrate. Bar graphs indicate the average of the corrected individual values ± propagated SD. Horizontal lines denote the upper and lower limits of the wildtype standard deviation. Three technical replicates from at least three independent experiments are shown.

Figure 4

Correlation of normalized variant transport. Surface corrected uptake values from Figure 3 are plotted against each other. Variant amino acid replacements are indicated by their respective letter, with “N” representing wildtype. (A) Comparison of ³[H]taurocholate to ³[H]estrone-3-sulfate transport. (B) Comparison of ³[H]taurocholate transport to ³[H]rosuvastatin uptake. (C) Comparison of ³[H]estrone-3-sulfate to ³[H]rosuvastatin uptake. Linear (Pearson) and rank order (Spearman) correlation calculations were determined and are reported in Table S1.

Figure 5

Substrate transport kinetics by wildtype and select 271 variants. Kinetic experiments were measured in HEK293 cells under initial linear rate conditions using increasing concentration of the respective substrates. Kinetics for wildtype (first column) were previously reported [8] and are shown here for visual comparison. NTCP variants N271C (second column), N271H (third column), and N271L (fourth column) were determined using (A) taurocholate, (B) estrone-3-sulfate, and (C) rosuvastatin. The transport capacity (V_max/K_m) of all variants and substrates are plotted in the fifth column. The Michaelis–Menten equation in GraphPad Prism 9 was used to determine the curves of best fit, and kinetic parameter results are reported in Table 1. Results were calculated from at least three independent experiments, each with 2–3 technical replicates, and are reported as the mean ± SD.

Figure 6

Visual representation of the correlation between kinetic values versus surface corrected initial transport for select N271 variants. Kinetic values, K_m (left column) and V_max (right column), for select N271 variants from Table 1 were plotted against their surface corrected initial uptake values (Figure 3) for each substrate (taurocholate, first row; estrone-3-sulfate, second row; and rosuvastatin, third row). Comparisons are meant to aid in visual observations of changes in kinetic values. Red letters correspond to wildtype (WT) and the amino acid substitutions in position 271.