Chaperones involved in protein folding

Chaperones involved in protein folding Overview of molecular chaperone families - distribution of chaperones in eukaryotes, archaea and bacteria Nascent-chain binding chaperones - Trigger Factor, NAC, Hsp70, prefoldin

Overview of chaperone families:Distribution 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:multigene families • 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)

“Clusters of Orthologous Groups of proteins” COG Homologues: genes that are related in sequence and function Orthologues: cross-species or cross-domain genes that are related in sequence and function Paralogues: homologous genes that were duplicated in the same organism http://www.ncbi.nlm.nih.gov/COG/xindex.html category: O Post-translational modification, protein turnover, chaperones *15--------qv--b-efghs-ujx-l- HslU [O] COG1220 ATP-dependent protease, ATPase subunit *48aomtpkzy--drbc-f-----j---- SpoVK [O] COG0464 ATPases of the AAA+ class 558---t---yqvdrbcefghsnujxilw ClpA [O] COG0542 ATPases with chaperone activity, ATP-binding domain *54aomtpkzyqvdrbcefghsnujxilw GroEL [O] COG0459 Chaperonin GroEL (HSP60 family) 226-------yqvdrbcefghsnujxilw GroES [O] COG0234 Co-chaperonin GroES (HSP10) 619-------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 *36aomtpkzyqvdrbcefg-s---x--- IbpA [O] COG0071 Molecular chaperone (small heat shock protein) 310aomtpk-yq----------------- GIM5 [O] COG1730 Prefoldin, molecular chaperone, beta class 9aomtpkz------------------- GIM1 [O] COG1370 Prefoldin, molecular chaperone, alpha class archaea bacteria yeast other categories: translation, transription, cell motility, ion transport, etc. etc.

Different sites of action Location of chaperone is very important: cytosol? membrane? organelle? extracellular? periplasmic? • e.g., clusterin binds large number of extracellular proteins • e.g., calnexin; must be near polypeptide entry? • ribosome-bound? • soluble? • associated with particular structures? • e.g., PapD/FimC is required for pilus folding/assembly • must bear sequence tag to target it there • chaperonin required for its own folding

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

Nascent-chain binding chaperone: TF • 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 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 Nascent-chain binding chaperone: NAC 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

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. NAC function: example experiment 77aaffLuc is the N-terminal 77 amino acids of firefly luciferase lacking an import signal Beatrix et al. (2000) J. Biol. Chem. 275, 37838.

NAC: a bona fide chaperone? • 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?

Nascent-chain binding chaperone: Hsp70 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

Hsp70 in de novo protein biogenesis • 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

Structure of Hsp70 chaperone • 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 ATPase domain (homology with actin, which also binds ATP) Polypeptide binding domain with bound peptide ‘substrate’

Substrate specificity of Hsp70 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

alkaline phosphatase Hsp70 binds short hydrophobic sequences catabolite activator protein influenza hemagglutinin • 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) tumour suppressor Rudiger et al. (1997) EMBO J.16, 1501

Bacterial DnaK functional cycle • 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

DnaJ (Hsp40) • 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

Nascent-chain binding chaperone: prefoldin 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

Prefoldin subunit structure 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 quaternary structure • most of surface is hydrophilic in character • inside tips of the coiled coils and ‘bottom’ of cavity display some hydrophobic character • 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

Prefoldin functional mechanism (a) PFD = prefoldin Pα = alpha subunit Pβ = beta subunit Siegert et al. (2000) Cell103, 621.

Prefoldin functional mechanism (b) • 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 (c)

Hsp70-like function of prefoldin? • 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

Chaperones involved in protein folding