Protein allosteric pathways are investigated in the imidazole glycerol phosphate synthase heterodimer in an effort to elucidate how the effector (PRFAR,formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) activates glutaminase catalysis at a distance of 25 Å from the glutamine-binding site. We apply solution NMR techniques and community analysis of dynamical networks, based on mutual information of correlated protein motions in the active and inactive enzymes. We find evidence that the allosteric pathways in the PRFAR bound enzyme involve conserved residues that correlate motion of the PRFAR binding loop to motion at the protein-protein interface, and ultimately at the glutaminase active site. The imidazole glycerol phosphate synthase bienzyme is an important branch point for the histidine and nucleotide biosynthetic pathways and represents a potential therapeutic target against microbes. The proposed allosteric mechanism and the underlying allosteric pathways provide fundamental insights for the design of new allosteric drugs and/or alternative herbicides.glutamine hydrolysis | protein networks | generalized correlation analysis | network theory A llostery is a fundamental property that allows for the regulation of function and dynamic adaptability of enzymes and proteins. Allosteric enzymes contain at least two distant binding sites, including the active site responsible for catalytic activity, which binds the substrate, and the allosteric site, which binds the effector and initiates the allosteric signal propagation to the active site. In V-type systems, substrate binding is not affected by the presence of the effector but if the effector is not bound, the allosteric protein is usually catalytically inactive (or poorly active), indicating that the effector binding is coupled to the kinetic and/or thermodynamic parameters of the biochemical reaction in the active site. Allosteric information transfer can range from large, enthalpically driven conformational changes to purely entropically driven motions or a combination of both enthalpic and entropic effects, but in each case the kinetic parameters of the catalyzed reaction at the substrate binding site are altered. At the heart of allosterism there is intramolecular thermodynamic coupling over long distances (>10 Å), between the active and allosteric sites. An important challenge for fundamental studies is the elucidation of the allosteric pathways that connect the two ligand-binding sites.In this work, we combine community network analysis based on molecular dynamics (MD) simulations and NMR studies of protein motion based on relaxation dispersion techniques and chemical shift titrations experiments to provide an atomistic description of allostery in the V-type allosteric enzyme imidazole glycerol phosphate synthase (IGPS) from the thermophile Thermotoga maritima (Fig. 1). IGPS is a tightly associated heterodimeric enzyme in which each monomer enzyme catalyzes a different reaction (1-3). The 23 kDa HisH enzyme is a member of the glutamine amidotransferas...

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.

Allosteric enzymes regulate a wide range of catalytic transformations, including biosynthetic mechanisms of important human pathogens, upon binding of substrate molecules to an orthosteric (or active) site and effector ligands at distant (allosteric) sites. We find that enzymatic activity can be impaired by small molecules that bind along the allosteric pathway connecting the orthosteric and allosteric sites, without competing with endogenous ligands. Noncompetitive allosteric inhibitors disrupted allostery in the imidazole glycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima as evidenced by nuclear magnetic resonance, microsecond time-scale molecular dynamics simulations, isothermal titration calorimetry, and kinetic assays. The findings are particularly relevant for the development of allosteric antibiotics, herbicides, and antifungal compounds because IGPS is absent in mammals but provides an entry point to fundamental biosynthetic pathways in plants, fungi, and bacteria. Graphical abstract *Corresponding Authors: ivan.rivalta@ens-lyon.fr.,

Allosterism is one of nature's principal methods for regulating protein function. Allosterism utilizes ligand binding at one site to regulate the function of the protein by modulating the structure and dynamics of a distant binding site. In this review, we first survey solution NMR techniques and how they may be applied to the study of allostery. Subsequently, we describe several examples of application of NMR to protein allostery and highlight the unique insight provided by this experimental technique.

Bile salt micelles can be employed as a pseudo-stationary phase in micellar electrokinetic capillary (MEKC) separations of chiral analytes. To improve MEKC separations of chiral analytes, a molecular level understanding of micelle aggregation in the presence of analyte is needed. Here, aggregation of sodium cholate has been observed by exploiting the presence of a model analyte molecule. The 31 P and 1 H nuclear magnetic resonance spectroscopy (NMR) chemical shifts of (R,S) 1,1′-binaphthyl-2,2′-diylhydrogenphosphate (R,S-BNDHP), a model analyte in chiral MEKC separations, are demonstrated to be very sensitive to the aggregation state of the bile salt sodium cholate. In addition to probing micellar aggregation, the NMR spectral resolution of enantiomeric species is also strongly correlated with chiral separations in MEKC. In this work, the aggregation of sodium cholate in basic solutions (pH = 12) has been observed over the concentration range 0-100 mM. The primary cmc was found to be 14 ± 1 mM for basic solutions of sodium cholate. In addition a primitive aggregate is clearly observed to form at 7 ± 1 mM sodium cholate. The data also show pseudo-cmc behavior for secondary aggregation observed in the regime of 50-60 mM cholate. Finally, the H5-H7 edge of BNDHP is shown to be sensitive to chirally selective interactions with primary cholate micelles.