BMB170c_2011_03_31_LECTURE

BMB170c_2011_03_31_LECTURE - BMB 170c Regulated Proteolysis...

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Unformatted text preview: BMB 170c Regulated Proteolysis 2 March 31, 2011 Regulatory Particle: Composition and assembly with the Core Particle Conserved architecture of proteasome Base: AAA+ ATPases Lid: Substrate recognition Walz et al, J. Struct. Biol 121: 19 (1998), Smith et al, Mol. Cell 20: 687 (2005) AAA+ ATPases form conserved hexameric rings ClpX 1UM8 1e94 Hexameric Ring Model of HslU Bochtler et al, Nature 403: 800 (2000) Crystal structures of HslU-HslV complex Bochtler et al, Nature 403: 800 (2000) Sousa et al, Cell 103: 63 (2000) Small Angle X-ray Scattering (SAXS) probes solution structure of macromolecules 5 2 q I (a.u.) 4 3 2 1 0 0 0.05 0.1 -1 q (A ) I - intensity; q = 4π sinθ / λ 0.15 Testing the crystal structures against solution models Sousa et al, Cell 103: 63 (2000) Testing the crystal structures against solution models Sousa et al, Cell 103: 63 (2000) Similar Organization of Archael Proteasome (PAN) How to dock a hexameric ring onto a Heptamer? Zhang et al, Mol Cell 34: 473 (2009) Eukaryotic 26S: Another layer of complexity Rpt - regulatory particle ATPase Rpn - regulatory particle non-ATPase – de-Ub activity – Ub binding activity Roles of the Regulatory Subunit 1. Substrate recognition 2. Gating into the proteolytic core 3. Protein unfolding and translocation using ATP hydrolysis Protein targeting to regulatory particles Matouschek, Curr. Opin. Struct. Biol. 13: 98 (2003) Damaged mRNA are C-terminally tagged with ssrA sequence Keiler et al, Science 271: 990 (1996) ssrA-tagged proteins are targeted to ClpXP for degradation ClpXP SspB ssr-tag sequence: AANDENYALAA ClpXP binds to ssrA-tagged substrates with the same affinity as ssrA peptide Keiler et al, Science 271: 990 (1996) Regulation of CP by the RP • Short peptides access CP and be rapidly digested • Longer peptides requires RP for gate-opening into CP (ATP hydrolysis not required) • Proteins require ATP-dependent unfolding by RP Thompson, Singh, Maurizi, JBC 269: 18209 (1994) Gating into the Core Particle Structure of yeast proteasome activator (PA26) Whitby et al, Nature 408: 115 (2000) Architecture of PA26-20S complex C-terminal loops of PA26 docks onto 20S α-subunits Whitby et al, Nature 408: 115 (2000) Activation loop induces a conformational change in αN 20S-PA26 20S Whitby et al, Nature 408: 115 (2000) PA26 opens a gate into the 20S particle 20S 20S-PA26 Whitby et al, Nature 408: 115 (2000) Protein Unfolding by AAA+ ATPases ClpAP mediates global unfolding of GFP Loss of fluorescence from folded GFP-ssr However, loss of fluorescence could be due to degradation of GFP This experiment alone cannot distinguish protein unfolding from degradation Weber-Ban et al, Nature 401: 90 (1999) ClpA Mediates Reversible Unfolding of GFP • ClpA binds and unfolds GFP • unfolded GFP is released into solution and refold • GroEL traps unfolded protein and prevents refolding Time (min) Trap = GroEL mutant that binds unfolded proteins irreversibly Weber-Ban et al, Nature 401: 90 (1999) ClpP drives irrversible GFP unfolding by degrading the protein Time (min) Weber-Ban et al, Nature 401: 90 (1999) GFP unfolding and degradation occur at the same rate Protein unfolding is rate-limiting for degradation Kim et al, Mol. Cell 5: 639 (2000) AAA+ ATPases are powerful protein unfolding machines GFP unfolding Rate acceleration = 107-fold Kim et al, Mol. Cell 5: 639 (2000) What’s the pathway for protein unfolding? Model 1: global unfolding from the core Model 2: local unfolding from N- or C-terminus : regions where unfolding initiates Rates of unfolding by ClpAP or proteasome do not correlate with global protein stability ΔG, Urea Denaturation (kcal/mol) τdeg ClpAP (min) τdeg proteasome (min) 4.4 ~ 10 ~ 25 >>4.4 >200 >200 Barnase 10.5 ~ 20 ~ 20 Barnase +barstar >>10.5 ~ 20 ~ 50 DHFR DHFR+MTX Lee et al, Mol. Cell 7: 627 (2001) Protein structure adjacent to degradation tag determines its ability to be degraded Circularly permuted mutant DHFR τdeg, –MTX τdeg, + MTX (min) (min) DHFR ~ 40 > 200 Pro25N ~ 10 ~ 10 Lys38N ~5 > 200 • protein unfolding initiates from the site of recognition tag • unfolding rate depends on the stability of local structure adjacent to the tag Lee et al, Mol. Cell 7: 627 (2001) Model for protein unfolding by AAA+ ATPases Mechanical unfolding by pulling on the tagged C- or N-terminus of target protein How is ATP hydrolysis used to unfold proteins? AAA+ ATPases in diverse cellular function White & Lauring, Traffic 8: 1657 (2007) AAA+ ATPases: Conserved Hexameric Ring NSF PAN HslU ClpX AAA+ ATPases: diverse function, conserved machinery NSF Clp ATP contributes to many different functions of the regulatory particle • ATP binding is required for AAA+ ATPase hexamer assembly • ATP binding is required for assembly of RP with CP • Binding of at least 4 ATPs is required for substrate recognition • Binding of at least 2 ATPs is required for pore opening • ATP hydrolysis is required for protein unfolding & translocation Engineer Titin mutants to systematically vary stability and unfolding kinetics Kenniston et al, Cell 114: 511 (2003) ATP consumption rate is constant during unfolding ~150 min-1 ~100 Unfolding is a stochastic process requiring iterative application of a constant force Kenniston et al, Cell 114: 511 (2003) How Many ATPase sites are required for AAA+ ATPase function? ClpX 1UM8 1e94 Proposal: concerted action of all six ATPase sites are required for protein unfolding 3 – 6 nucleotides bind HslU6 in different crystal structures HslU6 structure with six nucleotides: 1e94, 1g4a, 1g3i, 1g41, 1gh1, 1ht1, 1ht2, 1hqy, 1im2, 1ofh, 1ofi HslU6 structure with four nucleotides: 1do0 HslU6 structure with three nucleotides: 1do2 Engineer AAA+ motors with mixtures of active and inactive ATPases Two types of mutations to inactivate ClpX ATPase: E185Q (E): specifically blocks ATP hydrolysis R370K (R): blocks ATP hydrolysis and uncouples ATP binding from substrate binding (equivalent to empty site) Covalently mix wildtype (W) with E or R subunits Martin, Baker, Sauer, Nature 437: 1115 (2005) Diverse ATPase arrangements can support unfolding and translocation of substrates Titin degradation (unfolding + translocation + degradation) Titin degradation per wildtype subunit 0.05 -1 degradation rate (min ) -1 0.2 0.1 0.03 0.02 0.01 WEREER WWWRRR WWWWRR WWWWWR RWE/RWE WWR/WWR WEREER WWWRRR WWWWRR WWWWWR RWE/RWE 0 WWR/WWR WWW/WWW 0 0.04 WWW/WWW degradation rate (min ) 0.3 Degradation rate is proportional to the number of wildtype subunits in the hybrid hexamer Martin, Baker, Sauer, Nature 437: 1115 (2005) Asymmetric hexamer arrangement in ClpX Glynn et al, Cell 139: 744 (2009) Model: ATP-driven protein degradation by self-compartmentalizing proteases Pore loops of ClpX interacts with ssr-tag Pore loops of ClpX interacts with ssr-tag Disulfide x-linking experiments + ATPγS – nucleotides Martin, Baker, Sauer, Mol Cell 29: 441 (2008) Conserved tyr in pore loop Y → A mutation in Pore loop leads to weaker substrate binding and slippage Martin, Baker, Sauer, NSMB 15: 1147 (2008) Mutation of conserved tyr leads to enormous increases in the energy cost of unfolding Martin, Baker, Sauer, NSMB 15: 1147 (2008) Aromatic ring in pore loops grip substrates to drive unfolding and translocation Martin, Baker, Sauer, NSMB 15: 1147 (2008) Proteasome Inhibitors Peptide aldehyde inhibitors Ac-LLnLH (MG132) Slow binding inhibitors of chymotrypsin activity Vinitsky et al, Biochemistry 31: 9421 (1992) Specificity of aldehyde inhibitors β5 > β2 > β1 MG132 also inhibits serine and thiol-proteases Vinitsky et al, Biochemistry 31: 9421 (1992) Inhibition of all active sites is needed to block proteasome action Kisselev, Callard, Goldberg, JBC 281: 8582 (2006) Dipeptide Boronic acid inhibitors Bortezomib (Velcade) • empty p-orbital on Boron readily accepts nucleophilic attack by -OH Highly potent inhibitors of Proteasome Boronates are highly specific inhibitors for N-tm proteases Adams et al, Bioorg. Med. Chem. Lett. 8: 333 (1998) Crystal structure of Bortezomib with yeast 20S Groll et al, Structure 14: 451 (2006) The way nature does it… • non-covalent, reversible inhibitor • Selective proteasome inhibition in low nanomolar range • Inactive against other proteases Exploring multi-valency Groll, Huber, Moroder, J. Pept. Sci. 15: 58 (2009) 20S inhibition by bivalent inhibitors IC50 (µM) Groll, Huber, Moroder, J. Pept. Sci. 15: 58 (2009) ...
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This note was uploaded on 01/03/2012 for the course BI 170c taught by Professor List during the Fall '09 term at Caltech.

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