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Allosteric signaling

Allosteric signaling. Biochemistry Direct negative feedback Indirect feedback Cyclic processes. Allosteric mechanisms. Regulation by binding to a site other (άλλος allos ) than the catalytic site Multiple chemical states in complex molecules Concerted, 2 state model

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Allosteric signaling

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  1. Allosteric signaling • Biochemistry • Direct negative feedback • Indirect feedback • Cyclic processes

  2. Allosteric mechanisms • Regulation by binding to a site other (άλλος allos) than the catalytic site • Multiple chemical states in complex molecules • Concerted, 2 state model • Sequential, multi-state model • Product – mediated feedback control • Oxygen-hemoglobin binding

  3. Ligand induced conformation Deoxy-hemoglobin Oxy-hemoglobin Globin O2 allows pocket closure Small shift in helix One added AA interaction Heme-O2 Heme

  4. Concerted model • Equilibrium between distinct high and low affinity states • Multi-subunit molecules make a concerted or unified conformational change • Ligand binding increases the high affinity enzyme  cooperative binding • Monod et al., 1965 Apparent affinity of H/L mix Ligand 1 Low affinity state 0.8 0.6 Bound Ligand 0.4 0.2 High affinity state 0 -2 -1 0 1 2 Log (ligand)

  5. Sequential model • Equilibrium among multiple affinity states • Multi-subunit molecules undergo sequential conformational changes as each subunit binds ligand • Allows “Negative” cooperativity • Koshlind et al., 1966 Apparent affinity of H/L mix Ligand 1 High affinity state Low affinity state 0.8 0.6 Bound Ligand 0.4 0.2 0 -2 -1 0 1 2 Log (ligand)

  6. Glucose metabolism • Sequential phosphorylation of glocose • Symmetric cleavage to PEP & on to citric acid cycle • PFK is rate limiting Phospho-fructo-kinase PFK

  7. PFK • Activated by ADP, F1P • Inhibited by PEP (prokaryots) or citrate (eukaryots) Allosteric ADP binding site Active site No reactants With reactants PDB:3PFK PDB:4PFK

  8. PEP inhibits PFK PEP bound ADP bound Allosteric cofactor interacts with its own peptide chain and other subunit (green chain a; aqua chain b) PDB:4PFK PDB:6PFK PEP binding causes a large scale reorganization of four monomers (Concerted model)

  9. Allosteric homeostasis loop (PFK) • Homeostasis: stable feedback control Controller Plant General model Sensor PFK Glycolysis PFK increases F1,6P, glycolysis converts this to PEP, which inhibits PFK PEP PFK Glycolysis PFK increases F1,6P, glycolysis converts ADP to ATP, reducing ADP, which is an activator of PFK ADP

  10. Glucose storage • Extracellular glucose uptake • Phosphorylation by hexose kinase • Conversion to fructose 1,6,-bisphosphate • Storage in glyogen polymers • Conversion to UDP-glucose • Ligation by glycogen synthase PFK Phospho- fructose Fructose bisphosphate Glycolysis HK isomerase Glucose Phospho- glucose UDP glucose phosphorylase GS UDP- glucose Glycogen

  11. Glycogen synthase • Adds UDP-glucose to glycogen • Glucose-dependent • ATP-dependent Controller Plant Sensor Glycogen Synthase Glycogen Glucose-6-p G6P is both substrate (via UDP-G) and regulator. Nonlinear dependence of rate on G6P Rothman-Denes & Cabib, 1971

  12. G6P regulation of GS • Allosteric conformational change Without G6P With G6P Baskaran et al. 2010

  13. Cytoskeletal remodeling • Polymerizaton of actin filaments • Regulation of myosin contractility • Myosin Light Chain Kinase • Myosin Light Chain Phosphatase Focal adhesion RhoA ROCK MLP Cell motility

  14. Small GTPases • GTP is not usually a Pi donor • GTPases can be allosterically regulated allosteric regulators • GTPase timer • GAP switch • Guanine Activating Proteins (GAPs) • Facilitators of GTPase • Active of themselves • ie: GAPs may be allosterically regulated by GTP-GTPase EF-Tu, the eEF1 homolog

  15. Rho kinase, cytoskeletal remodeling • GTP holds RhoA domains close • Residues of now adjacent domains bind ROCK1 ROCK1 PDB:1FTN PDB:1S1C GDP-RhoA GTP-RhoA+ROCK1

  16. GDP-GTPase Inactive GTP hydrolysis GTP Exchange GTP-GTPase Active GTPase cycle • GTP hydrolysis limits time of activation • Many GTPase effectors are GAPs • eg: ribosome • Autoinhibitory, self-sensing controller • Many GTPases require GEFs • Less a sensor of [GTP] • More a communication method GAP GEF

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