Chaperones involved in folding (II)

1
Chaperones involved in folding (II)
8-1

• Post-nascent-chain binding chaperones
• Chaperonins (bacterial GroEL, eukaryotic CCT,
  archaeal thermosome)
• Small heat-shock proteins (Hsps)
• Hsp33

2
8-2
alpha-beta hemoglobin heterodimer
a
A chaperone for a-hemoglobin
alpha-hemoglobin stabilizing protein (AHSP)
b
3
GroEL/GroES chaperonin system
8-3

• GroEL forms homo-oligomeric toroidal complex
  dependent on GroES cofactor for function GroEL
  is essential for cell viability
• GroEL/GroES system may bind 10 of all bacterial
  cytosolic proteins but recent study shows
  only a portion of those are completely
  chaperonin-dependent
• Belongs to so-called Group I chaperonins which
  includes evolutionarily-related bacterial GroEL,
  mitochondrial Hsp60, and chloroplast Rubisco
  subunit-binding protein (Rubisco is most abundant
  protein on earth and requires chaperonin for
  folding)
• Functional mechanism is the best understood of
  all chaperonins

4
GroEL/GroES structure
8-4
crystal structure of E. coli GroEL/GroES

• GroEL has two stacked heptameric rings
  (equatorial domains form inter-ring contacts)
• GroES forms a single heptameric ring that binds
  co-axially to one GroEL ring (caps GroEL,
  preventing polypeptide exit or entry) binds only
  when GroEL in ATP state
• crystals structure without GroES has been
  solved, and with ATP-gamma S (non-hydrolyzable
  ATP analogue)
• mitochondrial chaperonin (Hsp60) is single-ring
  GroES from chloroplasts consists of a fused dimer

5
GroEL subunit structure
8-5

• chaperonins have 3 domains
• equatorial domain is the ATPase
• intermediate domain is a flexible hinge binding
  of ATP and GroES causes the apical domain to move
  upward and turn about 90 to the side
• apical domain is the polypeptide binding domain
  the binding site consists mostly of large, bulky
  hydrophobic residues
• (determined by mutation analysis)
• GroES binds to the polypeptide binding site
  displaces substrate into the cavity

6
Group I chaperoninfunctional cycle
8-6

• large conformational changes occur upon ATP and
  GroES binding cavity interior expands 2 fold,
  hydrophobic residues in apical domain turn away
  from the binding site and the interior becomes
  hydrophilic
• ATP --gt ADP transition is when folding takes
  place in the cavity when ATP is hydrolyzed, and
  ATP/GroES binds to trans ring (opposite the cis
  ring), GroES on cis ring dissociates and the
  polypeptide exits
• the polypeptide may not be folded upon exiting
  it could undergo another round of folding by
  either the same chaperonin, another chaperonin,
  or another chaperone

7
GroEL mechanism of action
8-7
1. Multivalent binding of substrate 2. Unfolding
of substrate (controversial) - evidence that
non-native protein is unfolded further upon
binding to GroEL and hydrolysis of ATP 3.
Combination of multivalent binding, unfolding may
re-direct folding intermediates to proper folding
pathway once inside hydrophilic chaperonin
cavity 4. Infinite dilution??? (cage model)
Paper presentation (next 3 slides) Farr et al.
(2000) Multivalent binding of nonnative substrate
proteins by the chaperonin GroEL. Cell 100,
561-573.
8
GroEL function single polypeptide
8-8

• N- and C-termini of GroEL (chaperonins in
  general) are buried inside the cavity
• construct is a fusion between all 7
  subunits--protein size is 400 kDa!
• the fusion protein assembles properly as judged
  by em reconstructions
• powerful tool for analyzing contribution of
  individual subunits to binding, etc.

9
GroEL function in vivo
8-9

• strain with wild-type GroEL under control of lac
  promoter (inducible with IPTG)
• without IPTG, strain growth arrests
• growth restored when covalent GroEL (fusion
  construct) is present this represents a growth
  of
• other constructs were tested in the absence of
  IPTG o represents no growth, represents
  very slow growth

10
GroEL function in vitro
8-10

• found that covalent GroEL was a bit less active
  at binding non-native proteins compared to
  wild-type GroEL mild protease treatment restored
  binding
• experiment binding of denatured protein to
  various constructs, isolation by SEC, and amount
  of bound proteins quantitated

conclusions gt require at least two or three
GroEL subunits for binding non-native proteins
these should preferably be in positions 1-3 or
1-4 (i.e., not immediately adjacent) gt
ability of GroEL/GroES to fold substrate
followed similar pattern (not shown)
11
Group II chaperonin system
8-11a
12
Group II chaperonin structure
8-11b
alpha-helical protrusion
GroES
side view of top ring
apical domain
apical domain
side view of bottom ring
intermediate domain
intermediate domain
thermosome side view
equatorial domain
equatorial domain
GroEL
thermosome
comparison of GroEL/ES complex (one subunit of
GroEL, one subunit of GroES) with single
thermosome (alpha) subunit
8 subunits per ring 4 alpha, 4 beta subunits
thermosome top view
13
8-11c
14
Group II chaperoninfunctional cycle
8-12

• open or closed states of thermosome (archaeal
  chaperonin related to CCT) were determined by
  SAXS experiments in the presence of nucleotides
  (ADP, ATP) or ADP in the presence of inorganic
  phosphate (PO4, or Pi) to simulate ADPPi
  transition state
• none of the studies have been carried out in
  presence of substrates assume open
  conformations can interact with substrate and
  closed state is involved in folding
• ATP?ADP transition somehow causes large
  conformational change

15
MODEL WHICH INCORPORATES THE SUBSTRATE
Douglas et al. Cell 2011
16
CCT-actin em reconstruction
8-13

• actin is composed of 4 subdomains, Sub1-Sub4
• hinge between domains Sub3-Sub4 and Sub1-Sub2 is
  flexible
• ATP binds in cleft between large and small
  domains
• actin cannot fold properly in the absense of ATP
• CCT-tubulin reconstruction also done tubulin
  makes more contacts with CCT subunits

17
Evolution of chaperonins, prefoldin and
actin/tubulin
8-14

• FtsA, actin homologue
• FtsZ, tubulin homologue

Evolution of eukaryotes

• CCT and prefoldin co-evolved essential for
  actin/tubulin biogenesis
• actin and tubulin are essential components of
  cytoskeleton
• cytoskeleton is required for large number of
  cell processes unique to eukaryotes, including
  intracellular movements, engulfment, etc. etc.
• hypothesis eukaryotes could not have evolved
  without CCT and prefoldin

18
Small heat-shock proteins
8-15

• found in all three domains of life, usually in
  multiple copies
• form large molecular weight complexes
• consist of three distinct domains

• can efficiently bind proteins on the aggregation
  pathway
• play important role in thermotolerance
  protecting proteins from aggregating under stress
  conditions
• cooperate with other chaperones (e.g., Hsp70) to
  renature proteins function, like that of
  prefoldin, is ATP-independent

19
Small Hsp crystal structure
8-16
- sizes of small Hsps range from 150 kDa to 800
kDa - smallest functional small Hsp is a
nonamer (trimer of trimer)

• crystal structure from Methanococcus jannaschii
  Hsp16 small Hsp (first archaeal genome to be
  sequenced) (wheat and ? Structures now also
  known)
• spherical shell composed of 24 subunits
• 2-, 3-, and 4-fold symmetry
• N-terminal domain (first 33 amino acids) were
  not resolved in the crystal structure these are
  likely to be flexible or disordered

20
Small Hsp surface view
8-17

• immunoglobulin domain fold (same as PapD/ FimC)
• dimer interface most extensive (building block)
• C-terminal region is exposed on surface
• N-terminal region faces interior of the oligomer
  (N-terminal region was not resolved in the
  crystal structure)

21
Wheat small HSP
8-18
End view
Side view
Dodecameric structure
van Montfort et al. Nature Structural Biology
(2001)
22
Hsp33 the redox chaperone
8-19

• exclusively bacterial induced during oxidizing
  (stress) conditions in the cell

Hsp33
oxidizing conditions (e.g., H2O2)
Hsp33/Hsp33 dimer
Hsp33

• domain-swapped dimer (active form) inactive
  monomer
• activation dependent on redox condition in cell
  under oxidizing (stress) conditions, disulfide
  bridges are formed and dimerization takes place
  conserved cysteines
• Hsp33 efficient in preventing protein
  aggregation in vitro

Jakob et al. (1999) Cell 96, 341.
23
Hsp33 substrate binding site
8-20

• two possible binding sites that are only
  available upon dimerization
• residues shown are highly conserved across
  bacterial Hsp33 proteins
• multivalent bindingagain?