Presentation on theme: "Molecular chaperones involved in degradation and other processes (I)"— Presentation transcript:
1 Molecular chaperones involved in degradation and other processes (I) 21-1Molecular chaperones involved in degradation and other processes (I)Chaperones involved in degradation- AAA ATPases: background- ClpA functional mechanism- katanin functional mechanism- archaeal PAN, VAT
2 AAA ATPases: functions 21-2AAA ATPases: functionsAAA is an acronymn for ATPases Associated with a variety of cellular ActivitiesAAA ATPases are conserved across all domains (archaea, bacteria, eukarya)the AAA module is one of the most abundant protein folds found in organisms; for example, yeast has ~50 proteins that have AAA modulesthe AAA module is present in many proteins that have highly diverse functionsprotein disassembly - ClpA, ClpX, etc.protein disaggregation - Hsp104protein unfolding (degradation) - accessories to protease complexes; sometimes they are joined to the protease (i.e., FtsH membrane protease)membrane trafficking - NSF dissociates the SNARE complex, which brings two membranes together to facilitate fusion in vesicle trafficking pathwaysmicrotubule-dependent processes: severing (katanin)organelle biogenesisDNA replication - regulates protein-DNA interactions by dissociating protein complexes (e.g., the ring-shaped E. coli clamp loader complex)recombination - e.g., E. coli RecA and related proteinsdynein - a microtubule-based motor required for chromosome locomotion, organelle transport, etc.
3 AAA ATPases: structures 21-3AAA ATPases: structuresAAA ATPases are almost always hexameric ring complexesthe AAA module is a domain that is found in a variety of proteins and can occur typically in one or two copiesit is the non-AAA module region of the proteins that confer specificity of functionthe module is about 200 amino acids in length, and contains Walker A and B motifs, which are nucleotide-binding folds; this fold is described as a P-loop-type NTP-binding sitethe AAA+ module is a subset of the AAA module but likely exhibits the same structurepartial NSF structureVale (2000) J. Cell Biol. 150, F13-19.
4 AAA ATPases: mechanism 21-4AAA ATPases: mechanismmost of the functions of AAA module-containing proteins can be ascribed to some type of binding and modulation of protein conformation (e.g., unfolding, disassembly)such a functional mechanism may be similar to that of the GroEL chaperonin, which may partially unfold proteins before sequestering them into the ‘folding chamber’AAA ATPases undergo conformational changes upon nucleotide binding/hydrolysis (whether it’s simply binding or hydrolysis may differ between different proteins and is a topic of debate)Using rings as a “molecular crowbar” (Vale, 2000)Two possible functions of AAA ATPases which both necessitate a significant conformational change in the AAA ATPase. (A) protein unfolding can also include ‘teasing apart’ protein complexes or aggregates. (b) motor activity may be a relatively specialized function of an AAA ATPase (dynein)
5 21-4bIn the cycle of ATP hydrolysis, release of ADP and phosphate (Pi) from dynein is associated with the power stroke. In the presence of ATP and vanadate (Vi), dynein forms a stable complex (ADP Vi-dynein), thought to mimic the ADP Pi state and hence the pre-power-stroke conformation of the motor. Its structure in the absence of nucleotide (apo-dynein) is thought to represent the post-power-stroke conformation
6 ClpA: an unfoldase (GFP fluorescence) Weber-ban et al. (1999) 21-5ClpA: an unfoldaseGFP11+ ClpA+ ClpP(GFP fluorescence)Weber-ban et al. (1999)Nature 401, 90-3Note: ssrA tag is recognized by ClpA and used to target proteins for degradation
7 ClpA: an unfoldase cont’d 21-6ClpA: an unfoldase cont’d‘trap’ denotes D87K mutant GroEL that can bind non-native proteins very effectively but does not release themATP-gamma-S is a non-hydrolyzable analogue of ATPresults show that ClpA can unfold GFP11 independent of the ClpP protease(GFP fluorescence)
8 ClpA: an unfoldase cont’d 21-7ClpA: an unfoldase cont’din experiments b and c, GFP11 is labeled with 35S-methionineCPK = creatine phosphate kinase, a component of an ATP-regenerating system that also includes phosphocreatine and ATPnote: in the absence of ATP, ClpA exists as a mixture of monomers/dimers; in the presence of ATP, it becomes hexameric+trap35S-GFP11(35S counts)+trap
9 ClpA: an unfoldase cont’d 21-8ClpA: an unfoldase cont’dHydrogen-deuterium exchange experimentIntroduce deuterated protein in normal H2O for some time, then monitor hydrogen exchange, which occurs when there are ionizable hydrogens (e.g., from COOH, NH3+) present in the protein (all proteins do). Monitoring of exchange is done by mass spectrometry, which can detect single dalton differences- exposed backbone and side chain amide protons (N-H) can exchange; those that are buried (‘protected’) cannot exchangedGFP11 (deuterated GFP11) was obtained by fully unfolding GFP11 in GuHCl and refolding it in deuterated waterClpA unfolds GFP11 to an extent comparable to its chaotrope-unfolded stateGuHCldH2OunfoldedGFP11GFP11dGFP11
10 Katanin: a ‘cellular samurai’ 21-9Katanin: a ‘cellular samurai’Dr. Lynne Quarmby in MBB studies KataninKatanin is part of the large AAA ATPase familyheterodimer consisting of 60 kDa microtubule-stimulated ATPase that requires ATP hydrolysis to disassemble microtubules, and 80 kDa subunit that targets the complex to the centrosome and regulates the activity of the 60 kDa subunitplays role during mitosis/meiosis in regulating microtubule length/dynamicskatanin catalyzes the severing of microtubulessevering (breaking apart) actin filaments is relatively easy, and involves dissociation of two adjacent subunits;breaking up microtubules, which consist of 13 protofilaments that form hollow tubes, is much hardermodel for actionmicrotubules act as a scaffold on which katanin oligomerizes after it exchanges ADP for ATPonce a complete katanin ring is assembled, ATP hydrolysis takes placeconformational changes in katanin that destabilizes the tubulin-tubulin contactsthe ADP-bound katanin has a lower affinity for tubulin and dissociates- shows that AAA ATPases are not necessarily associated with protein degradationbut function using a similar mechanism
11 Details: katanin function 21-10Hartman and Vale (1999) Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286,only ATP-boundform oligomerisescan monitor oligomerization of p60 katanin via FRET (Fluorescence Resonance Energy Transfer)Effect of microtubules on p60 oligomerization, ATPase, and microtubule severing activitiesNucleotide-dependent binding of p60 katanin to microtubulessedimentation gradientmixture of CFP-p60 and YFP-p60microtubule sedimentationassay - way to identify MAPs(microtubule-associated proteins)and to quantitate their bindingATPase: measurement of ATP hydrolysisFRET: oligomerisation ofCFP-p60 and YFP-p60Q: why the sudden decrease in ATPaseand oligomerisation at high MT conc’n?p60(E334Q) - does not hydrolyse ATP
13 katanin: summary of action 21-11katanin: summary of actionModel for microtubule severing by katanin. See text for detail of the mechanism. For simplicity, only a single protofilament of the microtubule is shown. T, DP, and D represent ATP, ADP + Pi, and ADP states, respectively. The relatively low affinity of katanin for nucleotide suggests that exchange of ATP for ADP would occur rapidly in solution. The conformational change is shown to occur with gamma-phosphate bond cleavage, although this could also occur as a result of gamma-phosphate release.
14 VAT: an archaeal AAA ATPase 21-12VAT: an archaeal AAA ATPaseVAT is an archaeal AAA ATPase that forms a homohexameric complexhomologue of p97, a protein that assists proteasome-dependent degradation in many contextsdisplays both refoldase and unfoldase activitiesdepending on Mg2+ concentration, it displays 10-fold differences in ATPase activityin low-activity state, it promotes the refolding of a denatured model substratein high-activity state, it promotes the unfolding of the same substrateStructure of VATFunction of VAT- N-terminal domain alone shows chaperone activity- ‘groove’ between two subdomains of N-terminal domain is mostly charged but might be substrate-binding site (speculative)- hypothesis: sustrate binding plus nucleotide-induced conformational change may yield both activitiesEM structure of VAT complex- from Coles et al. (1999) Curr. Biol. 9, 1158.NMR structure ofN-terminal domain
15 archaeal PAN keep in mind Benaroudj and Goldberg (2000) PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone. Nat. Cell Biol. 2,AAA ATPaseskeep in mind- PAN is very closely related to the eukaryotic AAA ATPases that are found at the base of the 19S regulatory complex- archaea do not possess the complete regulatory complex
16 PAN: another archaeal AAA ATPase 21-13PAN: another archaeal AAA ATPasePAN is an archaeal homohexameric complex that is evolutionarily related to the six different subunits of the eukaryotic proteasome AAA ATPase rpt2 proteins that form part of the regulatory particle and bind the core proteasomePAN is an acronymn for Proteasome Activating Nucleotidase, and as its name implies, it stimulates the activity of the proteasome and hydrolyzes nucleotides (ATP)PAN is not present in all archaeae.g., T. acidophilum lacks it but contains VAT,which may play an analogous functionhas typical molecular chaperone activity andit can unfold proteins for degradation by the proteasome- casein is a favoured substrate for degradation as it intrinsically adopts a proteolytically-sensitive conformationArchaeal proteasomes (150 ng) at a molar ratio of the complexes of 4:1 (subunit ratio of 2:1) with 3.4 µg of -[14C]casein in buffer E with 1 mM ATP (top line), with 1 mM AMP-PNP (middle line), with 1 mM ADP or control without nucleotide (lower line). The reaction mixture was incubated for various periods, and the generation of radioactivity soluble in 10% trichloroacetic acid was determined by liquid scintillation counting. [note: TCA precipitates proteins onto filters whereas smaller peptides or amino acids are not soluble]. Proteasomes alone, incubated with the same three nucleotides or without nucleotide, had similar activity as proteasomes incubated with PAN and without any nucleotide. PAN alone had no proteolytic activity when incubated under the same conditions