protein folding in the cell – 2

7/30/2019 Protein Folding in the Cell 2

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Protein Folding in the Cell 2

BIOC 212

Winter 2013

J ason C. Young


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Which amino acid side chains can form these interactions?

can the polypeptide backbone form any of these interactions?

hydrophobic, hydrogen bonds, Van der Waals

hydrophobic interactionsA, G, I, L, V, P, F, W, Y, M,

C, Q, N, T, R, K, E

hydrogen bondsD, E, R, K, H, N, Q, S, T, Y,C

Van der Waals interactions all

ionic bonds D, E, R, K, H

disulfide bonds C


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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 AnfinsenNobel 1972


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The Folding Process

Unfolded (denatured) domains have extended conformations with nosecondary or tertiary structure

Folding proceeds through intermediates that have increasingstructure, 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.


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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-nativeintermediates molten globule states

cytochrome B562 with Haem

METLNDTLKVVEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSP

EMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYHQKYR


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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 proteinsleads to insolubility

Incompletely folded proteins :

immediately after synthesis

ligand not available

previously native but unfolded by stress (eg. heat)

apo cytochrome B562 without Haem


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Folding Quality Control

Some folding intermediates canbe non-productive off-pathway

Cells have quality controlmechanisms to correct misfolding

Molecular Chaperones assist thefolding of proteins

Proteasomes degrade misfoldedor unfolded proteins


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Chaperones

Molecular Chaperones assist folding and prevent aggregation,without being part of the native state

Chaperones often recognize exposed hydrophobic patches offolding intermediates

Many chaperones are Heat Shock Proteins (HSP; eg. Hsp70 is the70 kDa HSP), highly expressed after stress

Chaperones are also essential under non-stress conditions


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Types of Chaperones

Some substrates require specific chaperones, or combinations ofchaperones

ATP-dependent chaperones actively promote folding

substrate binding and release are regulated by ATPase cycles

ATP-independent chaperones prevent aggregation and can catalyzesome folding steps

Cooperation between chaperones

cytosol endoplasmic reticulum


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Families of Chaperones

Different families of chaperone proteins use various biochemicalmechanisms protein folding toolkit

3 families of ATP-dependent chaperones, with different structuresand ATPase cycles

Chaperonins (Hsp60) Hsp70 Family Hsp90 Family


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Chaperone Families by Compartment

Co-evolution of chaperones with substrates

Chaperonins

(HSP60)HSP70 HSP90 small HSP prefoldin

calreticulin& calnexin

peptidyl-prolyl

isomerase

bacteria + + + + +

archaea + +

eukaryotes(human):

cytosol + + + + + +

ER lumen + + + +

mitochondria + + + +

ATP-dependent ATP-independent


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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


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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

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

T. thermophilus Hsp70

ATPase domain

peptide binding domain

ATP peptide


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HSP70 States

ATP-bound the ATPase domain pulls peptide binding domainopen, no substrate peptide binding

ADP-bound the peptide binding domain is separate and shut, fortight 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


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HSP70 Substrate Binding

Clamp onto short (7 amino acid) polypeptide segments that arehydrophobic 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

DnaKpeptide bindingdomain

PeptideNRLLLTG


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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 ATPand bind polypeptide

J domains do not bind substrate

Some DNAJ s also have separate substrate binding domains act as ATP-independent chaperones

J domain

Hsc70 ATPasedomain

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

contact site


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Types of DNAJ s

Type 1 DNAJ s 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 inyeast S. cerevisiae


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Type 1 DNAJ proteins have conservedarrangement of domains

J domain, substrate binding, dimerization

homodimers: 2 x 50 kDa subunits

bind short hydrophobic sequences

Type 1 DNAJ

substratebinding dimerizationJ domain

Type 1 DNAJ co-chaperonesyeast Ydj1 (Type 1)

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


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HSP70 NEFs

Nucleotide Exchange Factors (NEF) replace ADP with ATP andcause 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

ATPasedomain


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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


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HSP70 Proteins

human Hsc70 is constitutively expressed, Hsp70 is inducible

mechanisms are the same

also have forms in human mitochondria, ER lumen

human Type 1 HSP40: DnaJ A1, DnaJ A2

compartment HSP70DNAJ co-

chaperoneNEF

E. coli cytoplasm DnaK DnaJ GrpE

human cytosol Hsc70, Hsp70

DnaJ A1,DnaJ A2,

Hsp40& many others

Bag1, Bag2,

Hsp110& others


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How Does HSP70 Help Folding?

A DNAJ is always required, importance of NEF varies betweenHSP70s 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 DnaJ A2 and Hsc70

Type 1 DNAJ


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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 abar. How is Alice different from Bob and Carl? What will happen in

the bar?


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HSP70 Cycle

HSP70-ATP HSP70-ADP

Substrate Binding:

HSP70

DNAJ

Co-chaperoneinteraction with HSP70


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End of 2

Hartl et al. (2011) Molecular chaperones in protein folding andproteostasis. Nature 475, 324-332.

protein folding in the cell – 2

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