Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites

Abstract

For protein mutagenesis, a common expectation is that important positions will behave like on/off "toggle" switches (i.e., a few substitutions act like wildtype, most abolish function).However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: "rheostat" positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostat effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of "local" positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allostericallycoupled binding sites. Positions near the Ala binding site had rheostat outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

Identification of biochemically neutral positions in liver pyruvate kinase

“…hLPYK has many “functions.” We have developed a semi‐high throughput assay that can simultaneously characterize substrate affinity ( K a‐PEP for the substrate, phosphoenolpyruvate), binding of the allosteric inhibitor binding ( K ix ‐Ala for alanine), binding of the allosteric activator ( K ix ‐FBP for fructose‐1,6‐bisphosphate), allosteric coupling between the PEP and fructose‐1,6‐bisphosphate binding events ( Q ax ‐FBP ), and allosteric coupling between the PEP and Ala binding events ( Q ax ‐Ala ). By having a method that reports on many functions at once, we have found hLPYK to be a useful model to evaluate individual amino acid positions based on functional outcomes due to substitution 2,3 . In particular, our past studies have identified substitutions at nonconserved positions in and near the allosteric binding sites that modulated multiple functional parameters 2,3,5‐9 (Figure 1, green dots).…”

“…By having a method that reports on many functions at once, we have found hLPYK to be a useful model to evaluate individual amino acid positions based on functional outcomes due to substitution 2,3 . In particular, our past studies have identified substitutions at nonconserved positions in and near the allosteric binding sites that modulated multiple functional parameters 2,3,5‐9 (Figure 1, green dots).…”

“…Higher sequence entropies represent lower conservation, and the dotted lines and light gray box are used to highlight non‐conserved positions for which an alanine substitution leads to more than a 10‐fold change in at least one functional parameter. The overlaid green dots show previously characterized positions in the allosteric sites 2,3,5,13 and the magenta dots show seven of the positions substituted in this study (the other two positions included in the current study were not reported in the alanine scan) [Color figure can be viewed at wileyonlinelibrary.com]…”

“…Such studies often target positions that are conserved within a protein family, because conserved positions are likely to have key roles in function. The contributions of nonconserved positions have historically been overlooked, 1 although their substitution can also modify function 2,3 and cause disease 4 . We have been using human liver pyruvate kinase (hLPYK) to probe the functional roles of nonconserved protein positions.…”

“…We have been using human liver pyruvate kinase (hLPYK) to probe the functional roles of nonconserved protein positions. In our studies, substitutions at nonconserved positions in and near the allosteric binding sites modulated multiple functional parameters 2,3,5‐9 . To better understand how the nonconserved positions contribute to function, we wish to compare their biochemical, biophysical and structural characteristics to those of nonconserved positions that have little effect on function.…”