Nanometer Propagation of Millisecond Motions in V-Type Allostery

Self Cite

201

417

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...

Section: Resultssupporting

confidence: 81%

Allosteric pathways in imidazole glycerol phosphate synthase

Rivalta¹,

Sultan²,

Lee³

et al. 2012

Self Cite

201

417

“…Thus, whereas the intraprotein signaling may involve subtle structural changes, it may also involve propagated changes in flexibility (25). Precedent for such propagation includes millisecond motions for interdomain allostery (26), and subnanosecond motions for single domain allostery (27). This work expands the range of responses by suggesting that propagated changes in subnanosecond flexibility can assist interdomain communication within modular systems.…”

Section: Discussionmentioning

confidence: 81%

Stereospecific gating of functional motions in Pin1

Namanja

Wang

et al. 2011

164

Pin1 is a modular enzyme that accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs found in numerous signaling proteins regulating cell growth and neuronal survival. We have used NMR to investigate the interaction of Pin1 with three related ligands that include a pS-P substrate peptide, and two pS-P substrate analogue inhibitors locked in the cis and trans conformations. Specifically, we compared the ligand binding modes and binding-induced changes in Pin1 side-chain flexibility. The cis and trans binding modes differ, and produce different mobility in Pin1. The cis-locked inhibitor and substrate produced a loss of side-chain flexibility along an internal conduit of conserved hydrophobic residues, connecting the domain interface with the isomerase active site. The trans-locked inhibitor produces a weaker conduit response. Thus, the conduit response is stereoselective. We further show interactions between the peptidyl-prolyl isomerase and Trp-Trp (WW) domains amplify the conduit response, and alter binding properties at the remote peptidyl-prolyl isomerase active site. These results suggest that specific input conformations can gate dynamic changes that support intraprotein communication. Such gating may help control the propagation of chemical signals by Pin1, and other modular signaling proteins.allostery | protein dynamics | ligand dynamics | protein evolution P hospho-serine/threonine-proline (pS/T-P) motifs are signaling motifs within intrinsically disordered loops of cell cycle proteins (1). The imide bond between the pS/T and P residues can adopt either the cis or trans conformation. These conformations differ in their susceptibility to kinases and phosphatases that propagate the chemical signals governing the cell cycle. Accordingly, the cell must regulate the cis/trans populations of these pS/T-P motifs to ensure proper signal routing.In this context, the peptidyl-prolyl isomerase Pin1 has emerged as a critical regulator (2, 3). Pin1 is a reversible enzyme that catalyzes the cis-trans isomerization of the pS/T-P imide linkages (2, 3) of other signaling proteins, such as CDC25C, p53, c-Myc, NF-kB, cyclin D1, and tau (3). Pin1 engages when external events, such as S/T (de)-phosphorylation, change the cis-trans equilibrium. Pin1 then catalyzes the cis-trans isomerization, thereby accelerating the approach to the new equilibrium (1).Pin1 is a modular protein of 163 residues consisting of a WW domain (1-39) and a larger peptidyl-prolyl isomerase (PPIase) domain (50-163) (Fig. 1). A flexible linker connects the two domains. Both domains are specific for pS/T-P motifs (1). The WW domain serves as a docking module, whereas catalysis is the sole province of the PPIase domain. Earlier structural studies of Pin1 revealed conformational changes upon substrate interaction, thus motivating flexibility-function studies of Pin1 (4-6). Our previous NMR deuterium relaxation studies of Pin1 mapped the changes in flexibility of methyl-bearing side chains caused by interaction with an establi...

“…In fact, recent studies on dihydrofolate reductase (56), triosephosphate isomerase (57), ribonuclease A (58), HIV-1 reverse transcriptase (59), and imidazole glycerol phosphate synthase (60) show that slow microsecond to millisecond motions are severely dampened when these enzymes are inhibited (56,59,60) or when catalytically hindering mutations are introduced (57,58).…”

Section: Discussionmentioning

confidence: 99%

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy

Masterson

Shi

Metcalfe

et al. 2011

139

220

Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation.allostery | cooperativity | phospholamban | substrate recognition | intrinsically disordered proteins