Whole‐protein alanine‐scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism

Abstract

Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play “key” roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid side-chains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (activator) and alanine (inhibitor). Each non-alanine/non-glycine residue hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using limited numbers of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms.

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

“…This outcome is consistent with the predictions that several positions in the Ala binding site participate as "choke points" in networks of interactions 15 . Those predictions are further consistent with outcomes from a full protein alanine scan of hLPYK 13 .…”

“…To identify the appropriate width and number of histogram bins, the RheoScale calculator assessed the full experimental dataset for (i)minimal and maximal values for the parameter being assessed, (ii) average error of experimental measurements, and (iii) average number of variants available per position. Since completing our first study of "local" hLPYK rheostat positions 3 , we have obtained and collated a much larger dataset of hLPYK parameters and used them to refine RheoScale histogram analyses 12,13 . These include measurements for additional variants and multiple, independent measurements of wildtype hLPYK (Supplemental Information).…”

RheoScale: A tool to aggregate and quantify experimentally determined substitution outcomes for multiple variants at individual protein positions

“…The narrow range of change in K a‐PEP and presence of the gap is intriguing. Values within the gap are accessible by single substitutions as shown by the 483 “near‐dead” variant and results from a whole‐protein alanine scan (Tang & Fenton, ). Further analysis of the current dataset showed the gap and the 7‐fold distribution persisted when the two binding sites were analyzed separately from each other ().…”

Decomposing Dynamical Couplings in Mutated scFv Antibody Fragments into Stabilizing and Destabilizing Effects

“…27 Here dynamical coupling between all residue pairs is decomposed to establish a baseline for the propensity of dynamic allostery, and to obtain a higher order signature of protein dynamics to help elucidate stability and functional characteristics across protein mutants.…”