Protein Folding in the Cell 2

BIOC 212 Winter 2013 Jason C. Young

Which amino acid side chains can form these interactions?

A, G, I, L, V, P, F, W, Y, M, C, Q, N, T, R, K, E D, E, R, K, H, N, Q, S, T, Y, C all D, E, R, K, H C

hydrophobic interactions hydrogen bonds Van der Waals interactions ionic bonds disulfide bonds

can the polypeptide backbone form any of these interactions? hydrophobic, hydrogen bonds, Van der Waals

Protein Folding
Folding is thermodynamically favoured (negative G free energy) Folding can be spontaneous in principle, but assisted by different biological mechanisms Native structure is determined by the primary sequence

Christian Anfinsen Nobel 1972

The Folding Process

Unfolded (denatured) domains have extended conformations with no secondary or tertiary structure Folding proceeds through intermediates that have increasing structure, to the native state Molten Globule intermediates are close to native contain some secondary structure elements loosely packed and flexible compared to native state

Folding model of a single-domain protein. Daggett & Fersht (2003) Nature Reviews Mol Cell Biol 4, 497-502.

Native State
Structure is stabilized by hydrophobic contacts (blue) Some domains also require a ligand partner to be stable cofactor (Haem, steroid, etc), or another protein subunit Some domains are stable without ligand, but can still bind ligand reversibly Native state can be in equilibrium with near-native intermediates molten globule states
METLNDTLKVVEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSP EMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYHQKYR

cytochrome B562 with Haem

Folding Intermediates
Have some secondary structure, but tertiary structure incomplete Some but not all hydrophobic side chains are buried More of the polypeptide in flexible, disordered conformation Risk of aggregation interaction between different unfolded proteins leads to insolubility Incompletely folded proteins : immediately after synthesis ligand not available previously native but unfolded by stress (eg. heat)

apo cytochrome B562 without Haem

Folding Quality Control

Some folding intermediates can be non-productive off-pathway Cells have quality control mechanisms to correct misfolding Molecular Chaperones assist the folding of proteins Proteasomes degrade misfolded or unfolded proteins

Chaperones
Molecular Chaperones assist folding and prevent aggregation, without being part of the native state Chaperones often recognize exposed hydrophobic patches of folding intermediates Many chaperones are Heat Shock Proteins (HSP; eg. Hsp70 is the 70 kDa HSP), highly expressed after stress Chaperones are also essential under non-stress conditions

Types of Chaperones
Some substrates require specific chaperones, or combinations of chaperones ATP-dependent chaperones actively promote folding substrate binding and release are regulated by ATPase cycles ATP-independent chaperones prevent aggregation and can catalyze some folding steps Cooperation between chaperones cytosol endoplasmic reticulum

Families of Chaperones
Different families of chaperone proteins use various biochemical mechanisms protein folding toolkit 3 families of ATP-dependent chaperones, with different structures and ATPase cycles

Chaperonins (Hsp60)

Hsp70 Family

Hsp90 Family

Chaperone Families by Compartment

ATP-dependent
Chaperonins (HSP60) bacteria archaea eukaryotes (human): cytosol ER lumen mitochondria HSP70 HSP90 small HSP

ATP-independent
prefoldin calreticulin & calnexin peptidylprolyl isomerase

+ +

+ +

+ +

+ + +

+ +

+ + +

Co-evolution of chaperones with substrates

P
Rotation around backbone is slowed by large side chains Prolines rotate around backbone the slowest, because of extra covalent bond Slow proline rotation may limit the rate of folding Peptidyl-prolyl isomerases (PPIases) increase proline rotation and can speed up folding ATP-independent

HSP70 Family
HSP70 chaperones are 70 kDa monomers Highly conserved between species, and intracellular compartments Can have multiple functions in cells in addition to folding

peptide binding domain

ATPase domain T. thermophilus Hsp70

ATP peptide

Bertelsen et al. (2009) Proc Nat Acad Sci USA 106, 8471-8476

HSP70 States
ATP-bound the ATPase domain pulls peptide binding domain open, no substrate peptide binding ADP-bound the peptide binding domain is separate and shut, for tight substrate binding two-state mechanism is highly conserved

ATP-bound

ADP-bound / nucleotide-free

Kityk et al., Mol Cell 2012 Nov 1 Epub

Bertelsen et al., PNAS (2009) 106, 8471-8476

HSP70 Substrate Binding

Clamp onto short (7 amino acid) polypeptide segments that are hydrophobic and have an extended conformation There can be more than one Hsp70 binding sites in one polypeptide Many different unfolded proteins can be bound by Hsp70

Peptide NRLLLTG

DnaK peptide binding domain

DNAJ (HSP40) Co-chaperones

Co-chaperones are proteins which regulate chaperones DNAJ proteins have a J domain: J domains bind transiently to Hsp70, activate it to hydrolyze ATP and bind polypeptide J domains do not bind substrate Some DNAJs also have separate substrate binding domains act as ATP-independent chaperones Hsc70 ATPase
domain

contact site

J domain

Jiang et al. (2007) Mol Cell 28, 422-433

Types of DNAJs
Type 1 DNAJs have conserved substrate binding domains Type 2 have divergent substrate binding domains Type 3 do not bind substrate directly activate HSP70s for specialized functions Type 1 and some Type 2 act in folding

J domain proteins in yeast S. cerevisiae

Type 1 DNAJ
J domain substrate binding dimerization

yeast Ydj1 (Type 1)

Type 1 DNAJ co-chaperones

Type 1 DNAJ proteins have conserved arrangement of domains J domain, substrate binding, dimerization homodimers: 2 x 50 kDa subunits bind short hydrophobic sequences

Ramos et al. J Mol Biol (2008) 383, 155-166

HSP70 NEFs
Nucleotide Exchange Factors (NEF) replace ADP with ATP and cause release of bound polypeptide by Hsp70 In eukaryotes, there are 3 families of exchange factors: BAG, HspBP1 and HSP110 All open up the ATPase domain to release nucleotide

ATPase domain

HSP70 Cycle

DNAJ binds substrate J domain stimulates ATP hydrolysis by HSP70 substrate is transferred and bound by HSP70 in ADP state NEFs cause ATP re-binding and release of substrate
Hartl & Hayer-Hartl (2009) Nature Struc Mol Biol 16, 574-581

HSP70 Proteins
compartment E. coli cytoplasm HSP70 DnaK DNAJ cochaperone DnaJ DnaJA1, DnaJA2, Hsp40 & many others NEF GrpE Bag1, Bag2, Hsp110 & others

human cytosol

Hsc70, Hsp70

human Hsc70 is constitutively expressed, Hsp70 is inducible mechanisms are the same also have forms in human mitochondria, ER lumen human Type 1 HSP40: DnaJA1, DnaJA2

How Does HSP70 Help Folding?

A DNAJ is always required, importance of NEF varies between HSP70s and substrates Multiple, fast cycles of substrate binding and release Hsp70 binding prevents aggregation of a substrate, while release provides chances for it to fold Importance of the DNAJ? hypothesis: 2-point binding of substrate by DnaJA2 and Hsc70

Type 1 DNAJ

Names
An atheist, a priest and a rabbi go into a bar. (categories and their properties are important) Alice, Bob and Carl go into a bar. (names are less meaningful unless linked to categories) What is Alice? (a woman? an atheist? an alcoholic?) Alice is an atheist, Bob is a priest and Carl is a rabbi. They all go into a bar. How is Alice different from Bob and Carl? What will happen in the bar?

HSP70 Cycle

HSP70-ATP Substrate Binding: HSP70 DNAJ Co-chaperone interaction with HSP70

HSP70-ADP

End of 2
Hartl et al. (2011) Molecular chaperones in protein folding and proteostasis. Nature 475, 324-332.