Molecular chaperones involved in degradation and other processes I

1
Molecular chaperones involved in degradation and
other processes (I)
21-1
Chaperones involved in degradation - AAA ATPases
background - ClpA functional mechanism - katanin
functional mechanism - archaeal PAN, VAT
2
AAA ATPases functions
21-2

• 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.

3
AAA ATPases structures
21-3

• 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.
4
AAA ATPases mechanism
21-4

• 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 its
  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
21-4b
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
6
ClpA an unfoldase
21-5
GFP11 ClpA ClpP
(GFP fluorescence)
Weber-ban et al. (1999) Nature 401, 90-3

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

7
ClpA an unfoldase contd
21-6

• 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

(GFP fluorescence)
8
ClpA an unfoldase contd
21-7

• 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

trap
35S-GFP11
(35S counts)
trap
9
ClpA an unfoldase contd
21-8
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 GFP in GuHCl and refolding it in
  deuterated water
• ClpA unfolds GFP11 to an extent comparable to
  its chaotrope-unfolded state

10
Katanin a cellular samurai
21-9
Dr. Lynne Quarmby in Biological Sciences 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
11
Details katanin function
21-10
Hartman and Vale (1999) Microtubule disassembly
by ATP-dependent oligomerization of the AAA
enzyme katanin. Science 286, 782-785.
12
katanin summary of action
21-11
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.
13
VAT an archaeal AAA ATPase
21-12

• VAT is an archaeal AAA ATPase that forms a
  homohexameric complex
• homologue of p97
• 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
substrate-binding site (speculative) -
hypothesis sustrate binding plus
nucleotide-induced conformational change may
yield both activities
EM structure of VAT complex - from Coles et al.
(1999) Curr. Biol. 9, 1158.
NMR structure of N-terminal domain
14
PAN another archaeal AAA ATPase
21-13

• 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 proteolytically-sensi
tive conformation
Archaeal proteasomes (150 ng) at a molar ratio of
the complexes of 41 (subunit ratio of 21) with
3.4 µg of -14Ccasein 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