Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

Abstract

Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand, N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.allostery | NMR | community networks | millisecond motions T he underlying principles of allosteric regulation have been a focal point of enzymology and structural biology for decades as studies of allostery have evolved from two phenomenological models (1, 2) to recognize structural and conformational ensembles that define a broad range of enzymatic states (3-6). As a result, a great deal of emphasis has been placed on understanding dynamic contributions to allostery (7-12) and factors that enable small fluctuations in local conformations of enzymes to propagate information over large distances. A well-developed understanding of dynamic allostery opens up numerous avenues for insight into protein engineering (13,14), drug design (15), and mechanistic biochemistry (16)(17)(18)(19)(20)(21)(22). To establish universal principles for relevant enzymes, however, a link between dynamics and function must be made.The heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS) plays a crucial role in amino acid and purine biosynthesis in bacteria and other microorganisms by sequentially catalyzing the hydrolysis of glutamine (Gln) in its HisH subunit and the cyclization of the substrate and allosteric effector N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) in its HisF subunit (Scheme 1). IGPS is a V-type allosteric enzyme that is inactive unless PRFAR is bound, and communication between the active and effector sites is driven by the enhancement of conformational flexibility, namely, fluctuations taking place on the millisecond timescale (22-24). Based on NMR and computational evidence, these motions are believed to propagate from the PRFAR binding site to the glutaminase binding site, thereby disrupting a hydrogen bond between V51 and P10 of HisH. This H bond prevents formation of the oxyanion hole site necessary for glutamine hydrolysis, and its disruption allows IG...

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics

“…To gain insight into the allosteric signaling pathway within the full-length Cas9, 5 the protein was subjected to extensive computational analyses that are suitable for the detection of allosteric effects. [35][36][37][38] Figure 5. Allosteric signaling across HNH.…”

“…The allosteric pathway for information transfer has been investigated by employing correlation analysis and graph theory. [35][36][37][38] First, the generalized correlations (GCij), which capture non-collinear correlations between pairs of residues i and j, are computed. 65 In this correlation analysis, two variables (xi,xj) can be considered correlated when their joint probability distribution, A E , k C, is smaller than the product of their marginal distributions, ( E ) • A k C. The mutual information ( ) is a measure of the degree of correlation between E and k defined as function of A E , k C and ( E ) • A k C according to:…”

Engineered control of enzyme structural dynamics and function

“…(C)]. Amino acid substitutions at network positions in the HisH subunit led to changes in the microsecond‐millisecond timescale dynamics according to NMR relaxation dispersion spectroscopy, and decreased PRFAR‐enhancement of the HisF catalytic activity . Intriguingly, these groups have also found a small molecule inhibitor that binds at the HisH‐HisF interface also disrupts ImGPS motions coordinated by the proposed network .…”

“…112 The functional coordination of different active sites through amino acid interaction networks is exemplified by the histidine biosynthetic enzyme imidazole glycerol phosphate synthase (ImGPS). [115][116][117][118] ImGPS is a heterodimeric enzyme consisting of the HisH subunit, which catalyzes the hydrolysis of glutamine to produce ammonia, and the HisF subunit, which receives the ammonia and catalyzes its reaction with PRFAR (N'-[(5'phosphoribusyl}formimino]-5]aminoimidazle-4-carboxamide ribonucleotide). 119 The glutaminase activity of the HisH subunit is dependent on binding of PRFAR to the HisH subunit.…”

Residue-Level Allostery Propagates Through the Effective Coarse-Grained Hessian

“…There are many well established experimental techniques for characterizing allostery, including activity based assays to investigate non-Michaelis-Menten kinetic behavior, 19 H/D mass spectrometry, 20,21 as well as structural based approaches such as X-ray crystallography, 22 cryoEM, 23 and NMR. [24][25][26][27][28][29][30][31][32] Structural techniques can be used to identify residues that interact directly with or are structurally perturbed by the e↵ector molecule by comparing apo and e↵ector bound states of a protein. Identifying allosteric pathways is significantly more challenging.…”