Molecular chaperones involved in Molecular chaperones involved in degradation and other processes (I)degradation and other processes (I)

Chaperones involved in degradation- AAA ATPases: background- ClpA functional mechanism- katanin functional mechanism- archaeal PAN, VAT

21-1

AAA ATPases: AAA ATPases: functionsfunctions AAA is an acronymn for ATPases Associated with a variety of cellular Activities AAA 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 modules the AAA module is present in many proteins that have highly diverse functions

protein disassembly - ClpA, ClpX, etc. protein disaggregation - Hsp104 protein 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 pathways microtubule-dependent processes: severing (katanin) organelle biogenesis DNA 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 proteins dynein - a microtubule-based motor required for chromosome locomotion, organelle transport, etc.

21-2

AAA ATPases: AAA ATPases: structuresstructures AAA ATPases are almost always hexameric ring complexes the AAA module is a domain that is found in a variety of proteins and can occur typically in one or two copies

it is the non-AAA module region of the proteins that confer specificity of function

the 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 site the AAA+ module is a subset of the AAA module but likely exhibits the same structure

partial NSF structure Vale (2000) J. Cell Biol. 150, F13-19.

21-3

AAA ATPases: AAA ATPases: mechanismmechanism most 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)

21-4

In 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

21-4b

ClpA:ClpA: an unfoldase an unfoldase

Note: ssrA tag is recognized by ClpA and used to target proteins for degradation

(GF

P f

luor

esce

nce

)

Weber-ban et al. (1999)Nature 401, 90-3

21-5

GFP11+ ClpA+ ClpP

ClpA:ClpA: an unfoldase cont an unfoldase cont’’dd

‘trap’ denotes D87K mutant GroEL that can bind non-native proteins very effectively but does not release them ATP-gamma-S is a non-hydrolyzable analogue of ATP results show that ClpA can unfold GFP11 independent of the ClpP protease

(GF

P f

luor

esce

nce

)21-6

ClpA:ClpA: an unfoldase cont an unfoldase cont’’dd

in experiments b and c, GFP11 is labeled with 35S-methionine CPK = creatine phosphate kinase, a component of an ATP-regenerating system that also includes phosphocreatine and ATP

note: in the absence of ATP, ClpA exists as a mixture of monomers/dimers; in the presence of ATP, it becomes hexameric

(35S

cou

nts)

21-7

35S-GFP11

+trap

+trap

ClpA:ClpA: an unfoldase cont an unfoldase cont’’dd

dGFP11 (deuterated GFP11) was obtained by fully unfolding GFP11 in GuHCl and refolding it in deuterated water ClpA unfolds GFP11 to an extent comparable to its chaotrope-unfolded state

Hydrogen-deuterium exchange experiment

Introduce 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 exchange

21-8

GFP11GuHCl unfolded

GFP11dH2O

dGFP11

Katanin: Katanin: a a ‘‘cellular samuraicellular samurai’’

Katanin is part of the large AAA ATPase family heterodimer 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 subunit plays role during mitosis/meiosis in regulating microtubule length/dynamics

katanin catalyzes the severing of microtubules severing (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 harder

model for action microtubules act as a scaffold on which katanin oligomerizes after it exchanges ADP for ATP once a complete katanin ring is assembled, ATP hydrolysis takes place conformational changes in katanin that destabilizes the tubulin-tubulin contacts the 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

Dr. Lynne Quarmby in MBB studies Katanin

21-9

Details: Details: katanin functionkatanin function 21-10

Hartman and Vale (1999) Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286, 782-785.

can monitor oligomerization of p60 katanin via FRET (Fluorescence Resonance Energy Transfer)

Nucleotide-dependent binding of p60 katanin to microtubules

Effect of microtubules on p60 oligomerization, ATPase, and microtubule severing activities

p60(E334Q) - does not hydrolyse ATPp60(E334Q) - does not hydrolyse ATP

sedimentation gradient

microtubule sedimentationassay - way to identify MAPs(microtubule-associated proteins)and to quantitate their binding

microtubule sedimentationassay - way to identify MAPs(microtubule-associated proteins)and to quantitate their binding

ATPase: measurement of ATP hydrolysisFRET: oligomerisation ofCFP-p60 and YFP-p60

Q: why the sudden decrease in ATPaseand oligomerisation at high MT conc’n?

ATPase: measurement of ATP hydrolysisFRET: oligomerisation ofCFP-p60 and YFP-p60

Q: why the sudden decrease in ATPaseand oligomerisation at high MT conc’n?

mixture of CFP-p60 and YFP-p60mixture of CFP-p60 and YFP-p60

only ATP-boundform oligomerises

katanin: katanin: summary of actionsummary of action21-11

Model 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.

VAT: VAT: an archaeal AAA ATPasean archaeal AAA ATPase VAT is an archaeal AAA ATPase that forms a homohexameric complex homologue of p97, a protein that assists proteasome-dependent degradation in many contexts displays both refoldase and unfoldase activities

depending on Mg2+ concentration, it displays 10-fold differences in ATPase activity in low-activity state, it promotes the refolding of a denatured model substrate in high-activity state, it promotes the unfolding of the same substrate

EM structure of VAT complex- from Coles et al. (1999) Curr. Biol. 9, 1158.

NMR structure ofN-terminal domain

Structure of VAT Function 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 activities

21-12

archaeal PANarchaeal PANBenaroudj and Goldberg (2000) PAN, the proteasome-activating nucleotidase from archaebacteria, is a protein-unfolding molecular chaperone. Nat. Cell Biol. 2, 833-839.

AAA ATPases

keep 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

PAN: PAN: another archaeal AAA ATPaseanother archaeal AAA ATPase PAN 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 proteasome PAN 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 archaea

e.g., T. acidophilum lacks it but contains VAT,which may play an analogous function

has typical molecular chaperone activity andit can unfold proteins for degradation by the proteasome

Archaeal 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

- casein is a favoured substrate for degradation as it intrinsically adopts a proteolytically-sensitive conformation

21-13