Overview of molecular chaperone families

- distribution of chaperones in eukaryotes, archaea and bacteria

Nascent-chain binding chaperones- Trigger Factor, NAC, Hsp70, prefoldin

Chaperones involved in protein Chaperones involved in protein folding folding

Eukaryotes Archaea Bacteria- - Trigger Factor

NAC NAC -Hsp70 system [Hsp70 system] Hsp70 system

prefoldin prefoldin -chaperonins (group II) chaperonins (group II) chaperonins (Group I)

small Hsps small Hsps [small Hsps]Hsp90 - [Hsp90]

AAA ATPases AAA ATPases AAA ATPases- - SecB- - [PapD/FimC]

Hip, Hop, Bag, clusterin, cofactors A-E, calnexin,

calreticulin, etc. etc.- -

Overview of chaperone families:Overview of chaperone families:DistributionDistribution

not all molecular chaperone families are present in the three domains of life; some are highly specialized and are found in just one domain

eukaryotes have evolved not only more different families of chaperones, but typically have more members (e.g., Hsp70, small Hsps, prefoldin, etc.)

related to diversity of processes? (eukaryotes have organelles, greater diversity of cell functions)

must perform comparitive studies, e.g., with genome of the microsporidian Encephalitozoon cuniculi, 2.9 Mb. Amitochondriate, parasitic; cause of severe infections

bacteria and archaea do have chaperone multigene families

potential overlap in function? (e.g., Hsp70 in same/different compartments)

replacement of function by other chaperone families (e.g., prefoldin)

Overview of chaperone families:Overview of chaperone families:multigene familiesmultigene families

15 --------qv--b-efghs-ujx-l- HslU [O] COG1220 ATP-dependent protease, ATPase subunit

48 aomtpkzy--drbc-f-----j---- SpoVK [O] COG0464 ATPases of the AAA+ class

5 58 ---t---yqvdrbcefghsnujxilw ClpA [O] COG0542 ATPases with chaperone activity, ATP-binding domain

54 aomtpkzyqvdrbcefghsnujxilw GroEL [O] COG0459 Chaperonin GroEL (HSP60 family)

2 26 -------yqvdrbcefghsnujxilw GroES [O] COG0234 Co-chaperonin GroES (HSP10)

6 19 -------y---rbcefghs-ujx-l- HtpG [O] COG0326 Molecular chaperone, HSP90 family

70 -o-tp--yqvdrbcefghsnujxilw DnaJ [O] COG0484 Molecular chaperones (contain Zn finger domain)

7 -------------ce---s-uj---- CbpA [O] COG2214 Molecular chaperones, DnaJ class

36 aomtpkzyqvdrbcefg-s---x--- IbpA [O] COG0071 Molecular chaperone (small heat shock protein)

3 10 aomtpk-yq----------------- GIM5 [O] COG1730 Prefoldin, molecular chaperone, beta class

9 aomtpkz------------------- GIM1 [O] COG1370 Prefoldin, molecular chaperone, alpha class

category: O Post-translational modification, protein turnover, chaperones

archaeayeast

bacteria

“Clusters of Orthologous Groups of proteins”

Homologues: genes that are related in sequence and functionOrthologues: cross-species or cross-domain genes that are related in sequence and functionParalogues: homologous genes that were duplicated in the same organism

http://www.ncbi.nlm.nih.gov/COG/xindex.html

other categories: translation, transription, cell motility, ion transport, etc. etc.

COGCOG

Location of chaperone is very important:

cytosol? membrane? organelle? extracellular? periplasmic?

ribosome-bound? soluble? associated with particular structures?

e.g., calnexin; must be near polypeptide entry?

must bear sequence tag to target it there chaperonin required for its own folding

e.g., clusterin binds large number of extracellular proteins

e.g., PapD/FimC is required for pilus folding/assembly

Different sites of actionDifferent sites of action

chaperones can co-localize with: other chaperones protein degradation machinery different substrates etc.

Example:- misfolded proteins may end up in aggresomes (e.g., CFTR)- aggresomes contain various molecular chaperones, including Hsp70 and Hsp40, as well as proteasome components

This can potentially cause problems:- researchers expressed mutant CFTR- they then expressed mutant GFP that is normally broken down- saw GFP fluorescence (green) in the cytosol (i.e., it wasn’t degraded)- has implications for proteins that aggregate in cell and cause diseases

Co-localizationCo-localization

Trigger Factor (TF)

- most effective peptidyl prolyl isomerase (PPIase)

- behaves as a conventional molecular chaperone, i.e., can bind non-native proteins

- ribosome-bound (interacts with RNA in the 50S ribosome subunit, but some of it is cytosolic)

- interacts with large fraction of nascent polypeptides (as determined by cross-linking)

- only occurs bacteria (where it is ubiquitous), although other eukaryal/archaeal proteins have FKBP domains

- deletion is not lethal(!) However, deletion is lethal when knock out bacterial Hsp70, which also binds nascent chains

-crystal structure suggests that it forms a ‘pocket’ for chains exiting the ribosome

(recall the ‘crouching Dragon’ structure presented in class)

• how do the chaperone binding site and PPIase cooperate?

• what is the exact nature of the polypeptide binding site?

Nascent-chain binding chaperone: Nascent-chain binding chaperone: TFTF

Nascent polypeptide Associated Complex (NAC)

- eukaryotic protein consists of alpha and beta subunits; archaea have only beta subunit

- as with TF, bound to ribosome

- does not contain domain resembling a PPIase

Primary function:

- prevents inappropriate targeting of nascent polypeptides by SRP

- if ER signal sequence is present, SRP binds it, causes translation arrest, and docking occurs; co-translational insertion of protein then takes place, and the sequence is cleaved

- if ER sequence is not present, NAC prevents SRP from binding to the nascent chain

- evidence suggests it may help targeting to mitochondria

Nascent-chain binding chaperone: Nascent-chain binding chaperone: NACNAC

Beatrix et al. (2000) J. Biol. Chem. 275, 37838.

Fig. 8. NAC complex, but not the individual subunits, prevent inappropriate interaction of SRP with signal-less chains on ribosomes. High salt-stripped 77aaffLuc RNCs (ribosome nascent chains) obtained by in vitro translation in rabbit reticulocyte lysate, and carrying the photo-cross-linker (TBDA-modified lysine-tRNA), were incubated first with excess SRP, then with the individual NAC subunits, bovine NAC, or recombinant NAC as indicated. Samples were irradiated and analyzed by SDS-PAGE and fluorography. Bovine NAC (lane 6) and the reconstituted recombinant NAC (lane 5) both successfully competed with SRP for interaction with a signal-less chain on the ribosome. But neither alpha-NAC (lane 3) nor beta-NAC (lane 4) alone could prevent SRP from interacting with the signal-less nascent chain on the ribosome.

77aaffLuc is the N-terminal 77 amino acids of firefly luciferase lacking an import signal

NAC function: NAC function: example experimentexample experiment

If NAC is present at the polypeptide exit tunnel, and generally binds nascent chains (except when it is displaced by SRP), could it act as a molecular chaperone?

Is NAC functionally equivalent to Trigger Factor except for the fact it’s not a prolyl isomerase?

NAC: NAC: a a bona fidebona fide chaperone? chaperone?

Found in nearly all compartments where protein folding takes place:

- cytosol of eukaryotes (Hsp70) and bacteria (DnaK)

- mitochondria (mt-Hsp70)

- chloroplast (cp-Hsp70)

- endoplasmic reticulum (BiP)

- in yeast and nematodes, there are at least 14 different Hsp70’s

One surprising exception:

- not found in all archaea; this has been viewed as a paradox

- reason is that it has been shown to bind nascent polypeptides:

- it can be cross-linked to nascent chains in eukaryotes and bacteria

- another reason is that it is important for de novo protein folding

Nascent-chain binding chaperone: Nascent-chain binding chaperone: Hsp70Hsp70

Hsp70 is believed to bind and stabilize nascent polypeptides early in their synthesis--preventing misfolding and aggregation

Hsp70 binding and release, in an ATP-dependent manner, may help proteins fold to the native state OR Hsp70 may ‘transfer’ non-native proteins to other chaperones for folding (e.g., chaperonins)

Hsp70 is also important during cellular stresses (thermotolerance), and has numerous other functions in the cell apart from assisting de novo protein folding. It often works in collaboration with other chaperones, especially Hsp40

Hsp70 in Hsp70 in de novode novo protein biogenesis protein biogenesis

ATPase domain

(homology with actin

,

which also

binds ATP)

Polypeptide binding domain with bound peptide ‘substrate’

Structure of entire molecule (~70 kDa) has not been solved

flexible linkage between ATPase and peptide-binding domains, and different conformations of molecule possible

polypeptide-binding domain consists of beta-sheet scaffold; loops possess hydrophobic residues that contact peptide

domain also has an alpha-helical ‘lid’ that is regulated by the ATPase activity

Structure of Hsp70 chaperoneStructure of Hsp70 chaperone

Experiment

1. synthesize 13-mer peptides that overlap by 10 amino acids, based on actual protein sequences (spacer is Ala2)

- this covers entire protein sequence and any binding site

2. cross-link peptides to nitrocellulose membrane (automated)

3. add chaperone and allow binding to equilibrium

4. electro-transfer any Hsp70 bound to peptides onto membrane

5. probe membrane by Western blotting with specific antibody

6. screen 37 different proteins this way

7. obtain statistically significant information on binding motif

Substrate specificity of Hsp70Substrate specificity of Hsp70

alkaline phosphatase

catabolite activator protein

influenza hemagglutinin

tumour suppressor

Binding sites are either completely buried or partially shielded

Binding “ motif ” occurs every statistically occurs every 36 residues

Consistent with general binding affinity for nascent polypeptide chains (estimated at 20% or more)Rudiger et al. (1997) EMBO J. 16, 1501

Hsp70 binds short hydrophobic Hsp70 binds short hydrophobic sequencessequences

DnaJ (Hsp40 homologue) has affinity for unfolded proteins, and can deliver a substrate to DnaK

DnaK has fast on- and off-peptide binding rate when ATP is bound

DnaJ helps accelerate DnaK’s ATPase

DnaK has slow on- and off-peptide binding rate when in ADP conformation (i.e., it binds stably)

GrpE is a nucleotide exchange factor; it ‘opens’ up DnaK’s nucleotide binding site to help it release ADP and re-bind ATP

Released proteins may then be folded or might re-bind DnaJ/DnaK for another round of folding, or may interact with a chaperonin

Bacterial DnaK functional cycleBacterial DnaK functional cycle

Hsp40 may bind nascent polypeptides directly, passing these on to Hsp70

although it is a molecular chaperone in its own right, it seems to operate mostly in conjunction with Hsp70

there are numerous Hsp40 homologues in eukaryotes and bacteria; some are specific for the different Hsp70’s, and some actually modulate the function or localization of Hsp70’s

There also exists a number of additional chaperone cofactors that modulate the activity of Hsp70’s:

- e.g., Hip and Bag; these affect ATPase activity of Hsp70

in yeast, zuotin is an RNA-binding Hsp40 chaperone that is ribosome-bound; a cytosolic Hsp70 interacts with it to bind to nascent polypeptides

DnaJ (Hsp40)DnaJ (Hsp40)

Discovery

- a group performed a screen for yeast genes that were synthetically lethal in combination with a gamma-tubulin mutation

- found 5 genes that when disrupted, resulted in cytoskeleton defects

• actin: sensitivity to osmotic stress, latrunculin-A; disrupted actin filaments

• tubulin: sensitivity to benomyl; disrupted microtubules

- another lab independently purified a bovine protein complex containing 6 proteins that could bind unfolded actin and tubulin; the yeast complex was later purified and shown to possess the same 6 orthologous proteins as the bovine complex

Characterization

- synthetic lethality with various actin and tubulin mutants, as well as mutants involved in microtubule processes (i.e., cofactors A-E)

- may cooperate with cytosolic chaperonin (CCT) in actin and tubulin biogenesis

Nascent-chain binding chaperone: Nascent-chain binding chaperone: prefoldinprefoldin

Predicting coiled coils in proteins:

- a number of web-based programs are available

- rely on the repeating unit of the coiled coil

- a and d positions in a-g heptad repeat are usually hydrophobic

- the a and d positions form the apolar interface between the two helices; because of alpha helices normally have 3.6 residues/turn, the 3.5 residues/turn of the coiled coil induces a strain on the helix

Some coiled coils can have three or more helices

Prefoldin subunit structurePrefoldin subunit structure

Structure of archaeal prefoldin hexamer

oligomerization domain is a double beta-barrel structure

coiled coils are ~80A long and would be expected to behave independently

most of surface is hydrophilic in character

inside tips of the coiled coils and ‘bottom’ of cavity display some hydrophobic character

Prefoldin quaternary structurePrefoldin quaternary structure

Siegert et al. (2000) Cell 103, 621.

PFD = prefoldinPα = alpha subunitPβ = beta subunit

Prefoldin functional mechanism (a)Prefoldin functional mechanism (a)

Binding of prefoldin to unfolded proteins requires the multivalent interaction of the coiled coils

many other chaperones also bind in a multivalent manner

Prefoldin functional mechanism (b)Prefoldin functional mechanism (b)

Prefoldin functional mechanism (c)Prefoldin functional mechanism (c)

Prefoldin is found in all archaea but Hsp70 is not; those that have Hsp70 probably acquired it via lateral gene transfer

Mechanism of prefoldin is clearly different from that of Hsp70, but the overall function of each may be similar:

- both bind nascent polypeptides

- prefoldin can stabilize an unfolded protein for subsequent folding by chaperonin

(explanation in class)

- range of proteins archaeal prefoldin stabilizes is considerable: 14-62 kDa

Archaeal prefoldin (with 2 different subunits) may play a general role in protein folding whereas the eukaryotic chaperone (with 6 different subunits) may have acquired more specialized functions; this is seemingly the case for the eukaryotic chaperonin CCT, which has 8 different subunits compared to the archaeal chaperonin, which has 1 or 2 subunits, and the bacterial chaperonin (GroEL), which has 1 subunit

the presence of prefoldin may resolve the paradox that many archaea don’t have Hsp70, the otherwise ubiquitous molecular chaperone

Hsp70-like function of prefoldin?Hsp70-like function of prefoldin?