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-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostatic 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 allosterically-coupled binding sites. Positions near the Ala binding site had rheostatic 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.
Highlights
For protein mutagenesis, a common expectation is that important positions will behave like on/ off “toggle” switches
In mutagenesis studies that evaluate the contributions of important amino acid positions to protein function, our collective experience has led to a common expectation: For a given position, a few substitutions will result in wildtype-like function, but most substitutions will abolish function
We selected a broader range of positions for mutagenesis and in doing so, we identified another type of mutagenesis outcome: When these positions were substituted with various amino acids, a wide range of functional outcomes was obtained
Summary
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). Once rheostat positions were recognized to exist, we initiated studies to evaluate the prevalence of rheostat positions across a range of proteins with different, quantifiable functions[3] One such protein was human liver pyruvate kinase (hLPYK), a model system for allosteric regulation. Those previous studies included monitoring local effects; that is, we evaluated substitution effects on binding and allosteric regulation of the allosteric ligand with which the positions directly interacted (e.g., positions in the Fru-1,6-BP binding site were evaluated for effects on PEP/Fru-1,6-BP allosteric coupling) When scored for their rheostat character[3], most of the individual positions in hLPYK allosteric sites had modest rheostat scores for either binding of their allosteric ligand or for allosteric coupling between that effector and substrate binding
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