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 –Sequential, multi-state model Product – mediated feedback control Oxygen-hemoglobin binding

Ligand induced conformation Deoxy-hemoglobin Oxy-hemoglobin Globin Heme Heme-O 2 O 2 allows pocket closure Small shift in helix One added AA interaction

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 Low affinity state High affinity state Log (ligand) Bound Ligand Apparent affinity of H/L mix Ligand

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 Low affinity state High affinity state Log (ligand) Bound Ligand Apparent affinity of H/L mix Ligand

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

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

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

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

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 GlucoseGlucose-6P Phospho- fructose Fructose bisphosphate UDP- glucose Glycogen Glycolysis HK isomerase UDP glucose phosphorylase GS PFK

Glycogen synthase Adds UDP-glucose to glycogen Glucose-dependent ATP-dependent Glycogen Synthase (allosteric) Glycogen Synthase (catalytic) Glycogen Rothman-Denes & Cabib, 1971 Controller Sensor Plant G-6-P

G6P regulation of GS Allosteric conformational change Without G6PWith G6P Baskaran et al. 2010

Cytoskeletal remodeling Polymerizaton of actin filaments Regulation of myosin contractility –Myosin Light Chain Kinase –Myosin Light Chain Phosphatase Focal adhesion RhoAROCKMLPCell motility

Small GTPases GTP is not usually a P i 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

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

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 GEF GAP GDP-GTPase Inactive GTP-GTPase Active GTP Exchange GTP hydrolysis