Emerging maps of allosteric regulation in cellular networks

Abstract

Allosteric regulation is classically defined as action at a distance, where a perturbation outside of a protein active site affects function. While this definition has motivated many studies of allosteric mechanisms at the level of protein structure, translating these insights to the allosteric regulation of entire cellular processes - and their crosstalk - has received less attention, despite the broad importance of allostery for cellular regulation foreseen by Jacob and Monod. Here, we revisit an evolutionary model for the widespread emergence of allosteric regulation in colocalized proteins, describe supporting evidence, and discuss emerging advances in mapping allostery in cellular networks that link precise and often allosteric perturbations at the molecular level to functional changes at the pathway and systems levels.

Keywords: Allostery; Cellular signaling; Deep mutagenesis; Evolution of regulation; High-throughput biochemistry; Protein networks; Regulation.

Figure 1.. An integrative view of allostery.

(a) At the molecular level, allostery requires thermodynamic coupling between two distinct sites of a protein (left), for example an active site for binding of a small molecule ligand and a distal residue targeted by a post-translational modification (PTM, right). Perturbations at the regulatory site alter biochemical function at the active site. (b) In cellular networks, allostery enables independent regulatory mechanisms to control a central switch protein (left). Allosteric proteins can thus serve as network hubs, integrating signals from regulatory proteins (colored circles) and ligands (colored pentagons), and transmitting the signals to one or more downstream pathways. A key feature of network hubs is the selective activation of downstream pathways via independent regulation events (right) [32,62].

Figure 2.. A model for the widespread emergence of allostery in cellular networks.

(a) A general process by which proteins readily evolve new interactions and allosteric regulation was introduced by Kuriyan and Eisenberg [14]. Proteins that interact only weakly in solution can form complexes when colocalized due to an increase in effective concentration. Additional mutations that strengthen the interaction or establish allosteric regulation can evolve. (b) Inferred emergence of allosteric regulation of AurA kinase by TPX2, as described by [15]. (c) Inferred emergence of heterotetramerization and allosteric regulation in hemoglobin, as described by [16].

Figure 3.. A general approach for unbiased mapping of allosteric regulation by uniting molecular perturbations with systems-level profiling.

(a) Interrogation of allosteric regulation in cellular networks is achieved by systematically introducing molecular perturbations and measuring changes at each of the following scales: protein structure and dynamics (e.g. population of distinct conformations), biochemical activity (e.g. enzymatic rates or binding affinities), protein interaction networks (e.g. protein complex abundance and localization), and cellular phenotype (e.g. genetic interactions, cell morphology, and fitness). (b-d) Implementation of the approach for the eukaryotic small GTPase Ran/Gsp1, an allosterically regulated and multi-specific model protein switch, as described by [32]. (b) A series of 55 point mutations at 24 sites were introduced individually to the endogenous GSP1 gene in S. cerevisiae, targeting structurally characterized protein interaction interfaces outside of the nucleotide binding site. The unbiased approach identified four previously uncharacterized sites (shown in red) to be allosterically coupled to the nucleotide binding site conformation. Mutation of these sites changed conformational equilibria at the active site (c), thereby altering the kinetics of the GTPase cycle (d) and selectively perturbing pathways regulated by Gsp1 (e). (c) 1D ³¹P NMR spectra of Gsp1 variants bound to GTP showing that mutations at novel allosteric sites perturbed the relative populations of two conformations differing in the chemical shift of the terminal γ-phosphate of the bound GTP ligand (γ_GTPb). (d) Gsp1 mutations have differential effects on the GTPase switch kinetics. (e) The effects on kinetics selectively determined the effect on systems-level pathways as measured by genetic interaction (GI) profile correlations, which annotate the cellular effects of Gsp1 mutants by comparing them to the effects of knockdown of genes in various pathways: mutants with primarily reduced GTP hydrolysis (dotted arrow next to the orange GAP regulator in (d)) affected spindle assembly, mutants with primarily reduced nucleotide exchange rates (dotted arrow next to the blue GEF regulator in (d)) affected RNA modification, and all mutants altering cycling (hydrolysis and/or exchange) affected nuclear transport.