Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators

Abstract

1. Summary The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution NMR spectroscopy and molecular dynamics simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26 – 4900 fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Further, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity.

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

“…The V12A variant, however, displays 31 chemical shift perturbations above the significance cutoff, nearly the same as the 33 perturbations observed in WT IGPS, although the locations of shifts in each complex differ. Nonetheless, each ternary complex displays some degree of chemical shift perturbation due to PRFAR binding in locations spanning the entire HisF protein sequence, consistent with our previous findings of a widely dispersed allosteric network (22). (25), where communities f1′, f2′, f3′, and f4′ (symbolizing the PRFAR-bound enzymatic state) are colored light red, cyan, light green, and orange, respectively.…”

“…Important structural elements are labeled in each panel. Coupled with significant chemical shift perturbations (22), areas of broadening comprising the effector (PRFAR) ligand site, loop 1, the hydrophobic cluster, and the HisF/HisH interface are prevalent, although nearly every structural element of HisF is affected. A similar degree of chemical shift perturbation is observed in the V12A IGPS ternary complex, but the number of exchange-broadened resonances (30 in V12A vs. 67 in WT) decreases significantly.…”

“…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.…”

“…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)(23)(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 IGPS to conformationally sample the activated state. Recently, we have advanced these studies and demonstrated that activation of IGPS is dependent on the ability of allosteric effectors to stimulate domainwide motions in the HisF subunit and ligands that induce greater motion within the central (αβ) 8 barrel are more effective activators of glutaminase chemistry (22). NMR and computational community network studies proposed an optimal allosteric pathway through networks of HisF amino acid residues that link the PRFAR binding site and adjacent flexible loop 1 to residues comprising a central hydrophobic cluster essential for catalysis, as well as salt bridge residues at the dimer interface (25,26).…”