Protein regulation Protein motion

Transcription

1 Lecture 13 Protein regulation Protein motion Antoine van Oijen BCMP201 Spring /02 Section IV 04/09 Hands-on methods session / PS 4 due 1

2 Today s lecture 1) Mechanisms of protein regulation 2) Molecular switches Creating order in the chaos ( (David Goodsell) 2

3 Mechanisms of protein regulation - Localization/targeting - Covalent modifications - Environment - Effector binding Protein interaction domains (Petsko & Ringe) 3

4 Protein interaction domains - Typically between residue - C- and N-terminus close together to allow insertion into loop and stringing together of modules: Combinatorial richness (Petsko & Ringe) Protein interaction domains ( 4

5 Protein interaction domains See: for overview on different classes of protein interaction domains and their properties Localization / targeting - localization sequences ( tags ) - glycosylation - lipid modifications 5

6 Signal sequences Or bipartite localization sequences: e.g., nucleoplasmin (KR N 10 KKKK) localization patch Glycosylation Attachment of oligosaccharides to Asn, Ser, and Thr residues on protein surface Glc3Man9GlcNac2 precursor is attached in one piece (oligosaccharyl transferase) Added one-by-one monosaccharides (glycosyl transferase) 6

7 Glycosylation Most membrane and plasma proteins are glycosylated: - Recognition sites - Shielding of protein surface (proteases, non-specific interactions) E.g., antigenicity of HIV gp120 glycans HIV gp120 antibody (Scanlan et al., Nature (2007) Glycosylation determines blood type Blood group antigens on erythrocyte surface 7

8 The M6P pathway Mannose 6 phosphate Lipid modification Myristoylation (amide bond to C-14 f.a. chain) Palmitoylation (thioester bond to C-16 f.a. chain) (S-acylation) Prenylation (thioether bond to C-14 f.a. chain) E.g., targeting of GTPase Ras to cell membrane by lipid anchoring 8

9 Lipid modification GPI anchoring (Glycosylphosphatidylinositol) in mammalian proteins: Cell surface:nutrient uptake, cell adhesion, membrane signaling Reversible: released (often causing activation) by phospholipases Phosphorylation Transfer of terminal phosphate of NTP (usually ATP) to serine, threonine, or tyrosine residues (histidine and aspartate phosphorylation in prokaryotes) 9

10 Phosphorylation Sequential FAK activation (Mike Eck s lecture) Other post-translational modifications - Methylation - Acetylation - Nitrosylation - Sumoylation - Ubiquitination 10

11 Environment Redox environment: reducing inside cell, oxidizing outside (oxidation: loss of electron, reduction: gain of electrons) ---S-H H-S S-S--- Used to regulate oligomerization of extracellular proteins Environment ph: regulate ionization state of ionizable sidegroups; e.g., influenza fusion From: micro.magnet.fsu.edu 11

12 Environment ph: regulate ionization state of ionizable sidegroups; e.g., influenza fusion From: Harrison SC 2005 Mechanisms of regulation - Localization/targeting - Covalent modifications - Environment - Effector binding 12

13 Effector ligands Enzyme inhibition (competitive, noncompetitive), feedback inhibition Effector ligands Need for absolute, on/off behavior: cooperativity A A Protein A Not Cooperative A K 00A 00A + A K 0AA A A Protein A Cooperative A K 00A 00A + A τk 0AA τ >> 1 13

14 Effector ligands Need for absolute, on/off behavior: cooperativity A A Protein A Not Cooperative A K 00A 00A + A K 0AA A A Protein A Cooperative A K 00A 00A + A τk 0AA τ >> 1 Effector ligands Need for absolute, on/off behavior: cooperativity; e.g., hemoglobin 14

15 Effector ligands Allostery: binding of effector ligand causes structural changes Two models: sequential vs. concerted Allostery in oligomeric proteins is often cooperative Effector ligands Glycogen phosphorylase; glycogen glucose subunits Phosphorylation Ser14 activates enzyme AMP binding activates enzyme, ATP binding inactivates it 15

16 Protein switches Hydrolysis of NTP to NDP is coupled to control the on/off state of a process Two major classes: 1) GTPase ( G proteins ) 2) ATPase (motor proteins) Advantages using nucleotide triphosphates: - Large ΔG 0 (~ 30 kj/mole) - Switch state coupled to energy state of cell (NTP availability) Protein switches GTPase ATPase 16

17 Ras-Raf-MEK-ERK pathway Ras GTPase Ras protein: involved in variety of signaling pathways related to growth - Oncogene (mutations found in 20-30% of human tumors) - Coupled to membrane through prenylation 17

18 A protein switch based on hydrolysis state (Vetter et al., Science (2001)) Coordination of Mg 2+ in GTP-binding site (Pai et al., Nature 1989) Coordination of Mg 2+ in nucleotide-binding site a general feature of NTPases: - proper positioning of γ and β phosphates - maintain stability of nucleotide binding 18

19 Hydrolysis of GTP (Sondek et al., Nature (1994)) G tα, another small GTPase Hydrolysis of GTP (Sondek et al., Nature (1994)) A concerted mechanism for GTP hydrolysis (S N 2 type nucleophilic attack) 19

20 A switch mechanism (Vetter et al., Science (2001)) Cycling through hydrolysis states with GEF and GAP GEF: Guanine Exchange Factor GAP: GTPase Activating Protein 20

21 The GEF reaction, driving out the nucleotide (Vetter et al., Science (2001)) The GAP reaction, stimulating GTPase activity (Vetter et al., Science (2001)) GAP provides Arg that is missing in Ras, but necessary for fast GTP hydrolysis (transition-state stabilization) 21

22 ATP switches couple ATP hydrolysis to mechanical work Kinesin Myosin G protein ATP binding (Schwarzl et al., Biochemistry 2006) 22

23 Coupling unfavorable reactions with favorable ones A B C D K eq = [B] [A] = e["(g B "G A ) / RT] A + C B + D K eq = [D] [C] = e["(g D "G C ) / RT] K eq = [B + D] [A + C] = e["[(g B +G D )"(G A +G C )] / RT] As long as ΔG 0 C->D > ΔG 0 A->B, this reaction will occur spontaneously Hydrolysis of ATP is a frequently used favorable reaction: ATP ΔG=-30 kj/mol (~ 12 kt) ADP+P i Molecular motors Molecular motors couple nucleotide triphosphate hydrolysis (favorable) with mechanical work (unfavorable) Typical values: Stepsize 1-10 nm, force 1-10 pn: FΔx = x J ΔG 0,ATP->ADP = 30 kj/mol = 50 x J 23

24 Kinetic pathways of molecular motors Seitz, Surrey, EMBO Journal 2006 Multiple steps, intermediates Some load (force) dependent, some not Some ATP dependent, some not 24