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.

Allosterism is an essential biological regulatory mechanism. In enzymes, allosteric regulation results in an activation or inhibition of catalytic turnover. The mechanisms by which this is accomplished are unclear and vary significantly depending on the enzyme. It is commonly the case that a metabolite binds to the enzyme at a site distant from the catalytic site yet its binding is coupled to and sensed by the active site. This coupling can manifest in changes in structure, dynamics, or both at the active site. These interactions between allosteric and active site, which are often quite distant from one another involve numerous atoms as well as complex conformational rearrangements of the protein secondary and tertiary structure. Interrogation of this complex biological phenomenon necessitates multiple experimental approaches. In this article, we outline a combined solution NMR spectroscopic and computational approach using molecular dynamics and network models to uncover mechanistic aspects of allostery in the enzyme imidazole glycerol phosphate synthase.

Combining micellar electrokinetic capillary chromatography (MEKC) and nuclear magnetic resonance (NMR) experimentation, we shed light on the structural basis for the chirally selective solubilization of atropisomeric binaphthyl compounds by bile salt micelles comprised of cholate (NaC) or deoxycholate (NaDC). The model binaphthyl analyte R,S-BNDHP exhibits chirally selective interactions with primary micellar aggregates of cholate and deoxycholate, as does the closely related analyte binaphthol (R,S-BN). Chiral selectivity was localized, by NMR chemical shift analysis, to the proton at the C12 position of these bile acids. Correspondingly, MEKC results show that the 12α-OH group of either NaC or NaDC is necessary for chirally selective resolution of these model binaphthyl analytes by bile micelles, and the S isomer is more highly retained by the micelles. With NMR, the chemical shift of 12β-H was perturbed more strongly in the presence of S-BNDHP than R-BNDHP. Intermolecular NOEs demonstrate that R,S-BNDHP and R,S-BN interact with a similar hydrophobic planar pocket lined with the methyl groups of the bile salts, and are best explained by the existence of an antiparallel dimeric unit of bile salts. Finally, chemical shift data and intermolecular NOEs support different interactions of the enantiomers with the edges of dimeric bile units, indicating that R,S-BNDHP enantiomers sample the same binding site preferentially from opposite edges of the dimeric bile unit. Chirality 28:525-533, 2016. © 2016 Wiley Periodicals, Inc.

Bile salts are facially amphiphilic, naturally occurring chemicals that aggregate to perform numerous biochemical processes. Because of their unique intermolecular properties, bile salts have also been employed as functional materials in medicine and separation science (e.g., drug delivery, chiral solubilization, purification of single-walled carbon nanotubes). Bile micelle formation is structurally complex, and it remains a topic of considerable study. Here, the exposed functionalities on the surface of cholate and deoxycholate micelles are shown to vary from one another and with the micelle aggregation state. Collectively, data from NMR and capillary electrophoresis reveal preliminary, primary, and secondary stepwise aggregation of the salts of cholic (CA) and deoxycholic (DC) acid in basic conditions (pH 12, 298 K), and address how the surface availability of chirally selective binding sites is dependent on these sequential stages of aggregation. Prior work has demonstrated sequential CA aggregation (pH 12, 298 K) including a preliminary CMC at ca. 7 mM (no chiral selection), followed by a primary CMC at ca. 14 mM that allows chiral selection of binaphthyl enantiomers. In this work, DC is also shown to form stepwise preliminary and primary aggregates (ca. 3 mM DC and 9 mM DC, respectively, pH 12, 298 K) but the preliminary 3 mM DC aggregate is capable of chirally selective solubilization of the binaphthyl enantiomers. Higher-order, secondary bile aggregates of each of CA and DC show significantly degraded chiral selectivity. Diffusion NMR reveals that secondary micelles of CA exclude the BNDHP guests, while secondary micelles of DC accommodate guests, but with a loss of chiral selectivity. These data lead to the hypothesis that secondary aggregates of DC have an exposed binding site, possibly the 7α-edge of a bile dimeric unit, while secondary CA micelles do not present binding edges to the solution, potentially instead exposing the three alcohol groups on the hydrophilic α-face to the solution.