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

Abstract

Some protein positions play special roles in determining the magnitude of protein function: at such “rheostat” positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A (“I98X”). Despite being ~17 Å from the active site and far from subunit interfaces, substitutions at position 98 have rheostatic contributions to the apparent cooperativity (nH) associated with fructose‐1,6‐bisphosphate substrate binding and moderately affected binding affinity. Next, we crystallized representative I98X variants to assess structural consequences. Residues smaller than the native isoleucine (cysteine and serine) were readily accommodated, and the larger phenylalanine caused only a slight separation of the two parallel helixes. However, the diffraction quality was reduced for I98F, and further reduced for I98Y. Intriguingly, the resolutions of the I98X structures correlated with their nH values. We propose that substitution effects on both nH and crystal lattice disruption arise from changes in the population of aldolase A conformations in solution. In combination with results computed for rheostat positions in other proteins, the results from this study suggest that rheostat positions accommodate a wide range of side chains and that structural consequences manifest as shifted ensemble populations and/or dynamics changes.

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

“…Second, a subset of variants enhanced function relative to wildtype for both rheostat positions in NTCP. This observation is consistent with observations for rheostat positions in several other proteins [ 9 , 10 , 11 , 15 , 26 ].…”

“…In comparisons of LacI/GalR paralogs, the linker region that contains many rheostat positions [ 9 ] showed only local changes that did not propagate to the overall C α RMSD [ 25 ]. For human aldolase A, structures of rheostat variants showed very few changes among their crystallized conformations [ 26 ].…”

“…Thus, the neutral and rheostat positions must have some other underlying biophysical difference besides local structural tolerance to substitutions. One strong candidate is that substitutions at rheostat positions could alter functionally relevant dynamics [ 26 , 27 ]. Since dynamic conformational changes are key to transporter function, transporters—and multi-specific transporters especially—may contain a high density of rheostat positions.…”

Odd one out? Functional tuning of Zymomonas mobilis pyruvate kinase is narrower than its allosteric, human counterpart

“…First, we rationalized that since the only documented binding site on ZmPYK is the active site, nonconserved positions near the active site (sometimes referred to as “second shell” or “third shell”) might be more likely to include rheostat positions than distal regions of the protein. Second, since flexibility and dynamic changes have been associated with rheostat positions, 39,49 we assessed ZmPYK positions with a range of H/D exchange rates (Figure S3). Both strategies continued to sample positions with a range of phylogenetic scores (Table 2); in turn, the first set of phylogenetic positions extended the sampling of active site proximities and H/D exchange rates.…”

“…Thus, to avoid sampling bias in our studies, we have used sitedirected random mutagenesis at each targeted position. 15,39,51 This is critical for a long-term goal of building a library of rheostat positions from our combined studies. These substitution strategies were used in the current study to generate a range of variants at each of the chosen positions in ZmPYK.…”