Molecular chaperones involved in
degradation and other processes (I)
Chaperones involved in degradation
- AAA ATPases: background
- ClpA functional mechanism
- katanin functional mechanism
- archaeal PAN, VAT
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AAA ATPases: functions
 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.
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AAA ATPases: structures
 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 nucleotidebinding 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.
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AAA ATPases: mechanism
 most of the functions of AAA modulecontaining 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)
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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 Vidynein), thought to mimic the ADP Pi state and hence the prepower-stroke conformation of the motor. Its structure in the absence
of nucleotide (apo-dynein) is thought to represent the post-powerstroke conformation
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(GFP fluorescence)
ClpA: an unfoldase
GFP11
+ ClpA
+ ClpP
Weber-ban et al. (1999)
Nature 401, 90-3
 Note: ssrA tag is recognized by ClpA and used to target proteins for degradation
(GFP fluorescence)
ClpA: an unfoldase cont’d
 ‘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
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ClpA: an unfoldase cont’d
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(35S counts)
 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
+trap
35S-GFP11
+trap
 note: in the absence of ATP, ClpA exists as
a mixture of monomers/dimers; in the
presence of ATP, it becomes hexameric
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ClpA: an unfoldase cont’d
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
 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
GuHCl unfolded
GFP11
GFP11
dH2O
dGFP11
Katanin: a ‘cellular samurai’
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Dr. Lynne Quarmby in MBB studies Katanin
 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 degradation
but function using a similar mechanism
Details: katanin function
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Hartman and Vale (1999) Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286, 782-785.
only ATP-bound
form oligomerises
sedimentation gradient
Nucleotide-dependent binding
of p60 katanin to microtubules
mixture of CFP-p60 and YFP-p60
can monitor oligomerization of
p60 katanin via FRET
(Fluorescence Resonance Energy
Transfer)
microtubule sedimentation
assay - way to identify MAPs
(microtubule-associated proteins)
and to quantitate their binding
p60(E334Q) - does not hydrolyse ATP
Effect of microtubules on p60
oligomerization, ATPase, and
microtubule severing activities
ATPase: measurement of ATP
hydrolysis
FRET: oligomerisation of
CFP-p60 and YFP-p60
Q: why the sudden decrease in ATPase
and oligomerisation at high MT conc’n?
katanin: summary of action
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.
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VAT: an archaeal AAA ATPase
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 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
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 substratebinding site (speculative)
EM structure of VAT complex
- from Coles et al. (1999) Curr. Biol. 9, 1158.
NMR structure of
N-terminal domain
- hypothesis: sustrate
binding plus nucleotideinduced conformational
change may yield both
activities
archaeal PAN
Benaroudj and Goldberg (2000) PAN, the proteasome-activating
nucleotidase from archaebacteria, is a protein-unfolding molecular
chaperone. Nat. Cell Biol. 2, 833-839.
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
AAA ATPases
PAN: another 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 and
it can unfold proteins for degradation by the proteasome
- casein is a
favoured substrate
for degradation as it
intrinsically adopts
a proteolyticallysensitive
conformation
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
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