“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein

Abstract

Amino acid variation at “rheostat” positions provides opportunity to modulate various aspects of protein function – such as binding affinity or allosteric coupling – across a wide range. Previously a subclass of “multiplex” rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratory’s dataset of ∼4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) “single” rheostat positions that modulate only one functional parameter, (ii) “toggle” positions that follow textbook substitution rules, and (iii) “neutral” positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses – such as ConSurf – could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites.

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

“…Indeed, rheostat positions can be likened to a volume dial on a stereo, with substitutions dialing the functional “volume” either up or down. Furthermore, substitutions at some rheostat positions modulated only a single functional parameter (e.g., K d for binding ligand) whereas substitutions at others simultaneously modulated multiple parameters (e.g., substrate affinity, K d for binding allosteric ligand, and the magnitude of allosteric coupling between them) 1,3–7 …”

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

“…If >70% of substitutions maintain WT‐like function for all measured parameters, the position is assigned to the “neutral” category (Martin et al, 2020). If its set of substitutions samples ≥50% of the accessible functional range, the position is assigned to the “rheostat” category (Wu et al, 2019); this behavior has been observed for numerous positions in globular proteins (Bantis et al, 2020; Fenton et al, 2020; Hodges et al, 2018; Meinhardt et al, 2013; Ruggiero et al, 2021; Swint‐Kruse et al, 2021; Tang et al, 2017; Tang & Fenton, 2017; Wu et al, 2019). If >64% of substitutions abolish function, the position is assigned to the “toggle” category (Miller et al, 2017); this textbook substitution behavior is frequently observed at important, conserved positions in globular proteins.…”

“…In other proteins, the full tuneable range can be accessed by a variety of single substitutions (e.g., Tang & Fenton, 2017). Furthermore, in some globular proteins, 50%–100% of the tuneable, functional range can be sampled by a set of substitutions at an individual “rheostat” position (e.g., Bantis et al, 2020; Campitelli et al, 2020, 2023; Fenton et al, 2020; Page et al, 2022; Ruggiero et al, 2021; Swint‐Kruse et al, 2021; Wu et al, 2019). It is still unknown whether the existence of rheostat positions requires a classically‐folded protein structure or whether they can also be present in intrinsically disordered regions (IDRs).…”

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

“…If >70% of substitutions maintain WT-like function for all measured parameters, the position is assigned to the “neutral” category. 57 If its set of substitutions samples ≥50% of the accessible functional range, the position is assigned to the “rheostat” category; 2 this behavior has been observed for numerous positions in globular proteins 1–3 ; 5; 6; 9; 38; 58; 59 . If >64% of substitutions abolish function, the position is assigned to the “toggle” category; 60 this textbook substitution behavior is frequently observed at important, conserved positions in globular proteins.…”

“…In other proteins, the full tuneable range can be accessed by a variety of single substitutions (e.g., 1 ). Furthermore, site-saturating and semi-saturating mutagenesis (SSM and semiSM, respectively) of single positions revealed that, in some globular proteins, substituting individual "rheostat" positions can sample 50-100% of the tuneable functional range (e.g., [2][3][4][5][6][7][8][9] ). It is still unknown whether a classically folded protein structure is a pre-requisite for the presence of rheostat positions or whether they can also be present in intrinsically disordered regions (IDRs).…”