Roger Bartomeus
Jolita Jancyte
Helena Palma
Anna Perlas

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•
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Chaperones
Interactions between regions of the nonnative protein resulting into disordered complexes caused by self-association
Prevent large protein misfolding and aggregation without contributing conformational information
Alzheimer’s and Huntington’s disease
Cancer target
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from
Nascent Chain to Folded Protein. Science 295 (2002): 1852-58

Hsp70 System
 de novo protein folding
 ATPase activity
 Prevent or reverse intramolecular misfolding
 Block intermolecular aggregation
 Acts co- and posttranscriptionally

Hsp70 System
β-sandwich subdomain with a peptide binding cleft
α-helical latchlike segment
~44 kDa NH
2
-terminal ATPase domain ~27 kDa COOH-terminal peptide-binding domain

Hsp70 System
~7 residues (20-30 residues) target peptides hydrophobic (Leu and Ile)
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent
Chain to Folded Protein. Science 295 (2002): 1852-58

Hsp70 System
Regulated by:
NH
2
-terminal binds DnaK accelerating hydrolysis of ATP
COOH-terminal recognizes hydrophobic peptides
release of ADP rebinding of ATP

Chaperones
 Binds to ribosomes
 Interacts with proteins > 57 residues
 PPIase activity
 Overlapping function with Hsp70
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the
Cytosol: from Nascent Chain to Folded Protein. Science 295
(2002): 1852-58

Chaperones
 Nascent chain-Associated Complex
 Lacks PPIase domain
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent
Chain to Folded Protein. Science 295 (2002): 1852-58

Chaperonins
 Large double ring complexes
 ~800 kDa
 2 groups distantly related in sequence similar in architecture
GROUP I GROUP II
Eubacteria
Mitochondria and chloroplast
GroEL
GroES
Binding and release ATP regulated
Archaea
Eukaryotic cytosol
TRiC
GroES independent
Substrate captured through hydrophobic contacts

Group I
2 heptameric rings of 57 kDa subunits stacked back-to-back each subunit
– Equatorial domain (ATP binding site)
– Hingelike domain
– Apical domain
Substrate nonnative proteins up to 60 kDa
homoheptameric ring of ~10 kDa subunits
Mayer MP.Gymnastics of Molecular Chaperones.
Molecular Cell (2010) 39: 321-331

Group II
TCP-1 ring complex / CCT
8 heterogeneous subunits per ring that differ in their apical domain
Substrate: actin and tubulin

Prefoldin (PFD)
 GimC (genes involved in microtubule biogenesis complex)
 Hexameric ~90 kDa complex
 Encoded by 2 genes (archaea) or 6 genes (eukarya)
 ATP independent
 6 α-helical coiled-coil emanating from a double β-barrel
 Coiled coils partially unbound no interactions between them exposing hydrophobic amino acid residues
Archaea
2 PFD
α and 4 PFD
β
Binds unrelated proteins
Eukarya
2 α and 4 β subunits
Binds actin and tubulin

Prefoldin (PFD)
 2 structures from the pdb (the only crystallized structures of the whole prefoldin complex).
 Archaea
 1FXK: Methanothermobacter thermautotrophicus
–
–
–
Resolution: 2.3 Å
Missing residues: last 7 residues of the β chains
Labeled with Selenium (Selenomethionine)
 2ZDI: Pyrococcus horikoshii
–
–
Resolution: 3 Å
Missing residues:
●
●
●
α subunit: 1-3
β1 subunit: 1-4 and 111-117
β2 subunit: 1-9 and 111-117

Prefoldin structure
Side view of the prefoldin of M. thermautotrophicus

Prefoldin structure
Bottom view of the prefoldin of M. thermautotrophicus

70 Å
50 Å
Prefoldin structure
85 Å
50 Å
90 Å
30 Å

Prefoldin structure
Bottom of the beta-barrel core
- Patches near the ends of the coiled coils
Polar
Hydrophobic

Prefoldin structure
Bottom of the beta-barrel core
- Patches near the ends of the coiled coils
Non-Polar
Basic
Acidic
Polar

Prefoldin structure
N- and C- terminal regions form
α helices connected by one β hairpin linker
β hairpin 2 short β strands
Coiled coils
40 residues
12 turns

Prefoldin (PFD)
2 β hairpin
The extra one for the dimerization of α subunit monomers (residues 53-70)
Coiled coils
11 and 13 turns

Prefoldin classification

Architecture: Orthogonal Bundle (1.10)
Topology: Helix Hairpins (1.10.287)
Superfamily: (1.10.287.370)
Family: Prefoldin subunit alpha-like domain
Family: Prefoldin subunit beta-like domain

Prefoldin classification

Fold: Long alpha-hairpin
Superfamily: Prefoldin
Family: Prefoldin
Domain: Prefoldin alpha subunit
Domain: Prefoldin beta subunit

Prefoldin structure
 Eight-stranded
 Up and down
 Core filled with hydrophobic amino acids
β
α
β
β
β
α

Prefoldin structure

Prefoldin structure
Interchain interactions that stabilize the double β-barrel core of the complex
Mainly hydrogen bonds and salt bridges
The interactions between the two α subunits (chains C) are the most important contributors to the stability of the whole complex
(highest Complexation Significance Score)

α - α
Prefoldin structure

α - α
Prefoldin structure

α - β
Prefoldin structure

α - β
Prefoldin structure

β - β
Prefoldin structure

α - β
Prefoldin structure

α - β
Prefoldin structure

Assembly
A few months before publishing the first pdb structure of a prefoldin protein, the same authors performed several mass spectrometry assays with the purpose of unrevealing its quaternary structure
Collision results
• MtGimα
2
β
3
• MtGimβ
• MtGimα
2
β
2
• MtGimα
2
β
1
• MtGimα
1
β
2
2 MtGimα form a structural nucleus and MtGimβ subunits are organized around it
Fändrich M, Tito MA, Leroux MR, Rostom AA, Hartl FU, Dobson CM, et al. Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry. PNAS (2000) 97: 14151–14155

Prefoldin structure
 Bundle of α-helices wound into a superhelix


Opposite direction
Knobs-into-holes packing
 Heptad repeat (labeled a-g)
 3.5 residues per turn
 “a” and “d” are hydrophobic and form the helix interface a
140 Å d
22º
22⁰

Prefoldin structure
Core mean 6,290125 Å
Terminal mean 6,7275 Å

Archeal prefoldins
Structural similarity
RMSD 1.43
Sc 7.73
M. thermautotrophicus
P. horikoshii
Superimposition of the α subunits of our both templates: 1FXK and 2ZDI

Archeal prefoldins
Structural similarity
RMSD 1.01
Sc 8.31
M. thermautotrophicus
P. horikoshii
Superimposition of the β subunits of our both templates: 1FXK and 2ZDI

Interactions with unfolded proteins
Hydrophobic grooves
αLeu14
αVal131
αLeu11
αVal138
N
C

Interactions with unfolded proteins
βLeu15
βLeu12
βVal8
N
βLeu103
C
βLeu107

Interactions with unfolded proteins
Siegert R, et al. Structure of the Molecular Chaperone Prefoldin: Unique Interaction of Multiple Coiled Coil Tentacles with Unfolded Proteins. Cell
(volume 103 issue 4 pp.621 - 632) .

Flexibility of the tentacles
Superimposition of the β-hairpin of the three prefoldin subunits of M. thermautotrophicus
The coiled coils protrude from the core at different angles
Suggests flexibility

Flexibility of the tentacles
What we wanted to do
Assess the flexibility of the tentacles of MtPFD (pdb 1fxk)
Molecular Dynamics simulation (NAMD)
- Energy minimization
- Temperature: 338K (65 °C)
- Increase the temperature gradually from 100 to 338K
- Compare the mobility between the coiled coils and the beta core
- C
α
RMSD
- 1 ns simulation time at 338K
- Force field parameter: CHARMM22
- Periodic boundary conditions

Flexibility of the tentacles
What we succeeded in doing
 Solvation and neutralization of the system
 We managed to simulate 1.2 ps = 0.0012 ns
 Starting directly at 338K
 1 hour of computational time

Flexibility of the tentacles

Flexibility of the tentacles
What we tried
 Fix all the atoms except the C
α s
 Increase the time of each step
 Reduce the frequency of the output
 Simulate at lower temperature

Flexibility of the tentacles
What the guys from the article did
Fluctuation of PhPFD during MD simulation
Initial model
Simulated model
Ohtaki A, Kida H, Miyata Y, Ide N, Yonezawa A, Arakawa T, et al. Structure and Molecular
Dynamics Simulation of Archaeal Prefoldin: The Molecular Mechanism for Binding and
Recognition of Nonnative Substrate Proteins. J. Mol. Biol. (2008) 376: 1130–1141

Flexibility of the tentacles
What the guys from the article did
Fluctuation of PhPFD during
MD simulation
Ohtaki A, Kida H, Miyata Y, Ide N, Yonezawa A, Arakawa T, et al. Structure and Molecular Dynamics Simulation of Archaeal Prefoldin: The
Molecular Mechanism for Binding and Recognition of Nonnative Substrate Proteins. J. Mol. Biol. (2008) 376: 1130–1141

PFD-Actin interaction
3D reconstruction of the complex between PFD and unfolded different substrates
Archaeal PFD
Human PFD and unfolded actin
Archaeal PFD and unfolded actin
Martín-Benito J, Gómez-Reino J, Stirling PC, Lundin VF, Gómez-Puertas P, Boskovic J, et al. Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart.Cell press. (2007)15:101–110

Leu111
PFD-Actin interaction
PFD interaction sites (Experimental data)
Chains A and B:
C- terminal : Ala110-Leu111-Arg112-
Pro113- Pro114-Thr115-Ala116-Gly117-COOH
N -terminal : 8 – 10 first residues
Leu111
Gly112
Ala113
Gly114

PFD-Actin interaction
Unfolded actin interacts with PFD in a quasifolded conformation that requires the chaperone protection
Interaction with PFD
Interaction with CCT
Interaction with PFD and CCT
Martín-Benito J, Boskovic J, Gómez-Puertas P, Carrascosa JL, Simons CT, Lewis S a, et al. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT. The EMBO journal. 2002 Dec 2;21(23):6377–86

PFD-Actin interaction
2 types of PFD-CCT interaction
1. Asymmetric
2. Symmetric
PFD oligomers interact in each ring with 2 CCT subunits placed in a 1, 4 arrangement
Interactions between the
2 chaperones occurs at the level of the PFD tentacles outer regions and the inner surface of the chaperonin apical domains
Martín-Benito J, Boskovic J, Gómez-Puertas P, Carrascosa JL, Simons CT, Lewis S a, et al. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT. The EMBO journal. 2002 Dec 2;21(23):6377–86

Sequence Alignment
Sequence alignment between 3 archaeal α subunits and 2 eukaryotic ones (one coming from human and another from yeast)

Sequence Alignment
Sequence alignment between 3 archaeal β subunits and 2 eukaryotic ones (one coming from human and another from yeast)

Secondary Structure Comparison
Alignment of the predicted secondary structure of both human α subunits and pdb-extracted secondary structure of α subunits of both templates

Sequence Alignment
Alignment of the predicted secondary structure of the four human β subunits and pdb-extracted secondary structure of β subunits of both templates

Modelling human PFD
–
–
–

Modelling human PFD
Discarded

Modelling human PFD

Modelling human PFD

Modelling human PFD
–
–

Modelling human PFD

Modelling human PFD
β-subunits
RMSD 1.87
Sc 7.73
Dark blue : model
Light blue : phyre model
RMSD 1.94
Sc 6.80
Dark blue
Green
: model
: phyre model
Superimpositions for subunits 1 and 2 of both templates and the two models

Modelling human PFD
β-subunits
RMSD 2.95
Sc 5.95
Dark blue : model
Light blue : phyre model
RMSD 1.15
Sc 7.97
Dark blue
Green
: model
: phyre model
Superimpositions for subunits 4 and 6 of both templates and the two models

Modelling human PFD
α-subunits
RMSD 2.30
Sc 5.01
RMSD 1.73
Sc 6.04
Dark blue : model
Green : phyre model
Loops breaking helixes!
Red : model
Green : phyre model
Superimpositions for subunits 3 and 5 of both templates and the two models

Modelling human PFD
Loops breaking helixes!
First problems appear: not so good alignments caused some unrealistic features

Check the energy profiles
Subunit 1
Cyan : template 1FXK
Green : template 2ZDI
Magenta : model
Yellow : phyre model
Subunit 2

Check the energy profiles
Subunit 4
Cyan : template 1FXK
Green : template 2ZDI
Magenta : model
Yellow : phyre model
Subunit 6

Check the energy profiles
Subunit 3
Cyan : template 1FXK
Green : template 2ZDI
Magenta : model
Yellow : phyre model
Subunit 5

THREADING
Higher scores of THREADER for the human subunit 5 of prefoldin (an α subunit)

THREADING
Highest score corresponded to 1I36
It was meant to fail but we run modeller anyway…
Chain A

THREADING
… and, obviously, it did fail.

THREADING
Higher scores of THREADER for the human subunit 5 of prefoldin (an α subunit)

THREADING
The second better score was that of 2cgp, a gene activator
It is not really surprising since coiled-coils are DNA binding motifs
But still it is obvious that with that template we will not obtain a relying model at all

THREADING
Higher scores of THREADER for the human subunit 5 of prefoldin (an α subunit)

THREADING
Unfolded tails again!
So we just run modeller again with the same template but the alignment obtained by threader

THREADING
Threader Modeller
Putative models of human’s prefoldin subunit 5
Phyre

Check the energy profiles: conclusions
 The models made based on homology with the templates and using the threader alignments are always longer than the one obtained with phyre
 This means that phyre is cutting the sequence to avoid unaligned regions and subsequent unfolded segments
 This strategy allows phyre to obtain structures with a better energy profile although it does not contemplate the entire human sequence
 Because of that, we must use complete model
in order to obtain a

Paralogy
Sequence similarity
Alignment of the two human α subunits

Paralogy
Sequence similarity
Alignment of the four human β subunits

Curious case: Skp
 17 kDa
 Homotrimeric periplasmic chaperone
 Can interact with outer-membrane proteins
 β-barrel body surrounded by 3 C-terminal α helices
 N- and C- terminal ends are part of the trimerization core
No sequence homology with PFD
No structural homology with PFD but structural similarity
CONVERGENT EVOLUTION

Skp
C
N
N C

Skp: resemblance of prefoldin
 Circular Permutation is an evolutionary process
 Prefoldin and Skp structures are quite similar as a result of convergent evolution
 So, this is NOT a case of circular permutation because there is NO common ancestor but the final result is the same
 Neither STAMP or XAM allow us to do disordered superimpositions
 We searched the web for programs that could handle circular permutation

Superimposition of Skp and PFD
Blue : Skp
Red : prefoldin
1-45
10-50
RMSD 6.20
63-103
59-103

Superimposition of Skp and PFD
RMSD 2.87
Sc 2.57
Superimposition by STAMP
Purple : Skp
Green : PFD

Superimposition of Skp and PFD

Superimposition of Skp and PFD
Red : PFD
Green : Skp
RMSD 1.43
68 superimposed residues
RMSD 1.78
52 superimposed residues

Superimposition of Skp and PFD
Red : Skp
Green : PFD

Superimposition of Skp and PFD

Superimposition of Skp and PFD

Conclusions
 Chaperones are important to prevent misfolding and aggregation being Hsp70 system one of the most representative groups
 Prefoldin has a unique quaternary structure formed by 6 α-helical coiled-coil protruding from a double β-barrel principally stabilized thanks to hydrogen bonds and salt bridges
 Eukaryotic prefoldin has a more specific function that archaeal prefoldin interacting only with actin and tubulin
 Sequence and secondary structure similarity between the archaeal and eukaryotic prefoldins suggests that a model of the human prefoldin can be built from archaeal templates
 Several models can be built using either modeller, phyre or threader but neither of them is achieving a complete model that covers the whole human sequence. Using
Rosetta could be a possible approximation to solve this problem
 Skp and prefoldin are a good example of convergent evolution

Bibliography
 Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein.
Science (2002) 295: 1852-58
 Martín-Benito J, Gómez-Reino J, Stirling PC, Lundin VF, Gómez-Puertas P, Boskovic J, et al. Divergent
Substrate-Binding Mechanisms Reveal an Evolutionary Specialization of Eukaryotic Prefoldin Compared to
Its Archaeal Counterpart. Elsevier Structure 15 (2007) 101–110
 Ohtaki A, Kida H, Miyata Y, Ide N, Yonezawa A, Arakawa T, et al. Structure and Molecular Dynamics
Simulation of Archaeal Prefoldin: The Molecular Mechanism for Binding and Recognition of Nonnative
Substrate Proteins. J. Mol. Biol. (2008) 376: 1130–1141
 Siegert R, Leroux MR, Scheufler C, Hartl FU, Moarefi I. Structure of the Molecular Chaperone Prefoldin:
Unique Interaction of Multiple Coiled Coil Tentacles with Unfolded Proteins. Cell (2000) 103: 621–632
 Walton TA, Sousa MC. Crystal Structure of Skp, a Prefoldin-like Chaperone that Protects Soluble and
Membrane Proteins from Aggregation. Molecular Cell (2004) 15: 367–374
 Sahlan M, Zako T, Tai PT, Ohtaki A, Noguchi K, Maeda M, et al. Thermodynamic Characterization of the
Interaction between Prefoldin and Group II Chaperonin. J. Mol. Biol. (2010) 399: 628–636
 Fändrich M, Tito MA, Leroux MR, Rostom AA, Hartl FU, Dobson CM, et al. Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry. PNAS (2000) 97: 14151–14155

PEM
1) The chaperon prefoldin is present in: a) Archaea b) Eukarya c) The two previous ones are correct d) Bacteria e) All of the previous answers are correct
2) Select which one of the following statements is false: a) The GroEL-GroES complex is present in eubacteria b) Chaperonins need ATP for both binding and release of their substrates c) Skp is a prefoldin-like chaperone found in archaea d) Chaperones mainly interact with nonnative structures by hydrophobic interactions e) Trigger Factor binds to ribosomes
3) Prefoldin has: a) Two beta subunits and four alpha subunits b) Four beta subunits and four alpha subunits c) Two beta hairpins and four alpha helices d) Four beta hairpins and two alpha helices e) Two beta barrels and six tentacle-like coil-coiled helices
4) About the interactions between prefoldin subunits: a) Interactions between beta subunits are the most important ones b) Interactions between alpha and beta subunits are the most important ones c) The previous ones are correct d) Interactions between alpha subunits are the most important e) All of the previous answers are correct

PEM
5) Select the incorrect one: a) Archaeal prefoldin binds to several nonnative structures b) Eucaryotic prefoldin binds to actins and tubulins c) Prefoldin has several hydrophobic residues at the terminal segments of its tentacles thus allowing its interaction with nonnative structures d) Bacterial prefoldin needs cooperation with chaperonins e) The jellyfish-like shape of prefoldin provides high flexibility to the structure
6) About prefoldin and Skp is/are TRUE:
1.
Both prefoldin and Skp share the very same structure
2.
Skp and prefoldin similarities are a result of convergent evolution
3.
Skp chaperone is found in jellyfish, primates and humans
4.
N and C terminal ends of Skp chaperone are part of the trimerization core a) 1, 2 and 3 b) 1 and 3 c) 2 and 4 d) 4 e) 1, 2, 3 and 4
7) The archaeal prefoldin interacts with: a) Actin b) Tubulin c) The previous ones are correct d) Different unfolded archaeal proteins e) All the answers are correct

PEM
8) Prefoldin interacts with actin through: a) The specific regions of tentacle tips b) Exclusively polar regions c) Exclusively hydrophilic regions d) All the answers are wrong e) All the answers are correct
9) Select the TRUE answer about prefoldin: a) Superimposition of the β-hairpin of the 3 prefoldin subunits results in protruding coiled coils at different angles that suggests flexibility b) To study the flexibility of the tentacles is necessary a molecular dynamics simulation c) The previous ones are correct d) Tentacles are the most flexible part of the prefoldin chaperone e) All the previous statements are correct
10) What is true about the coiled-coil motif: a) It is a motif consisting of alpha-helices and beta-strands b) It is characterized by a heptad repeat in which the first (a) and fourth (d) residues tend to be hydrophobic c) Both previous statements are correct d) The hydrophobicity of the coiled-coil core helps to achieve the periodicity of the heptad repeat e) All previous statements are correct.

β - β
Prefoldin structure

To be informative, some mean of estimating the reliability of a multiple sequence alignment is required. T-coffee uses a method allowing the unambiguous identification of the residues correctly aligned. This method uses an index named CORE (Consistency of the Overall Residue Evaluation)
The CORE is obtained by averaging the scores of each of the aligned pairs involving a residue within a column. It is an indicator of the local quality of a multiple sequence alignment. Where q and r are two residues found aligned in the same column:

Given two residues q and r taken from two different sequences S1 and S2, one can easily measure the consistency between the alignment of these two residues and all the other alignments contained in the library by comparing the extended score of the pair q and r with the sum of the extended scores of all the other potential pairs that involve S1 and S2 and either r or q.

Imagine two residues R and S of two different sequences that are aligned in one position.
In the end, a pair of residues is highly consistent (and has a high extended weight) if most of the other sequences contain at least one residue that is found aligned both to R and to S in two different pair-wise alignments. A key property of this weight extension procedure is to concentrate information: the extended score of RS incorporates some information coming from all the sequences in the set and not only from the two sequences contributing R and
S. Extended scores are used like a position specific substitution matrix.

Class: Membrane and cell surface proteins and peptides
Fold: OmpH-like
Superfamily: OmpH-like
Family: OmpH-like
Domain: Periplasmic chaperon skp (HlpA)
Species: Escherichia coli [TaxId: 562]
Class: Alpha Beta
Architecture: 2-Layer Sandwich
Topology: Protein Binding, DinI Protein; Chain A
Homology: OmpH-like (Pfam 03938)