The co-PIs Loria and Batista from Yale will investigate allosteric pathways in the enzyme imidazole glycerol phosphate synthase (IGPS) from T. maritima, at the molecular level, with emphasis on the influence of small molecule modulators that bind to the IGPS allosteric interface and affect the molecular mechanisms that synchronize the enzyme catalytic activity with effector binding at the allosteric site. IGPS is ideally suited for studies of allostery since it is a protein heterodimer, composed of the HisH and HisF proteins, with most of the properties of classical allosteric enzymes, including an oligomeric structure, multiple ligand binding sites, multiple conformational equilibria in the absence of ligand, and the stabilization of specific protein conformations by ligands. It is a potential therapeutic target since it is not found in mammals and is found in bacteria as well as in some plants and fungi. In particular many plant pathogens and opportunistic human pathogens such as Cryptococcus, Candida, and Ajellomyces that infect immunocompromised individuals have an IGPS that is highly homologous to the S. cerevisiae and T. maritima enzymes. Additionally, it has recently been shown that gene knockouts of HisF from Acinetobacter and Burkholderia pseudomallei increase the susceptibility of the former to ?-lactam antibiotics and lessen the infectivity of the latter. However, the underlying allosteric mechanisms that could represent targets for drug discovery have yet to be established and will be explored by the proposed research program. The research hypotheses are: (i) allosterism involves motions of specific amino acid residues induced by PRFAR binding~ (ii) motions in HisF are transmitted to HisH and generate an active conformation of the HisH active site~ and (iii) motions communicating the active sites of HisF and HisH are affected by drug binding, or site-directed mutagenesis. The proposed methods combine Batista's computational modeling, including microsecond molecular dynamics simulations on the Anton supercomputer system from David E. Shaw Research, LLC at the National Resource for Biomedical Supercomputing of the Pittsburgh Supercomputing Center, network analysis, simulations of NMR spectra and computational drug screening, with Loria's state-of-the-art NMR relaxation techniques, quantifying the microsecond-to-millisecond conformational motions induced by drug or ligand binding with atomic resolution, mutagenesis studies, and isothermal titration calorimetry. The research program involves multiple cycles of an iterative approach where, in each cycle, allosteric pathways are explored through the analysis of differential motions probed by liquid-NMR relaxation methods and computation (MD and network analysis), obtaining valuable information on key amino acid residues and specific interactions responsible for transmitting structural or dynamical changes spanning the allosteric and active sites. The resulting insight provides guidelines for the next round of studies of mutants and modulators in a joint experimental and theoretical effort to elucidate the IGPS allosteric mechanisms as influenced by small molecule binding and site-directed mutagenesis.
State-of-the-art NMR techniques, site-directed mutagenesis and computational studies based on microsecond molecular dynamics/network analysis, drug screening and simulations of NMR spectra will explore the allosteric pathways in the enzyme imidazole glycerol phosphate synthase (IGPS) from T. maritima, a potential therapeutic target found in plant pathogens and opportunistic human pathogens.
|Lisi, George P; Manley, Gregory A; Hendrickson, Heidi et al. (2016) Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators. Structure 24:1155-66|
|Lisi, George P; Loria, J Patrick (2016) Solution NMR Spectroscopy for the Study of Enzyme Allostery. Chem Rev 116:6323-69|