Roger Bartomeus Jolita Jancyte Helena Palma Anna Perlas.

Chaperones

Prefoldin

Roger BartomeusJolita JancyteHelena PalmaAnna Perlas

INDEX• INTRODUCTION

– Chaperones– Hsp70 System– Chaperonins

• PREFOLDIN (PFD)– Archaeal PFD structure– Interactions– Conservation between archaeal

and eukaryotic PFD– Modelling human PFD– A curious case: Skp

• CONCLUSIONS

Chaperones

MISFOLDINGInteractions between regions of the nonnative protein resulting into disordered complexes caused by self-association

CHAPERONE FUNCTIONPrevent large protein misfolding and aggregation without contributing conformational information

Alzheimer’s and Huntington’s diseaseCancer target

Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein. Science 295 (2002): 1852-58

Eubacteria, eukarya and archaea

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

DnaK

Hsp70 System

~44 kDa NH2-terminal ATPase domain ~27 kDa COOH-terminal peptide-binding domain

DnaK

Hsp70 System

~7 residues (20-30 residues)

hydrophobic (Leu and Ile)target peptides


Regulated by:

DnaJ NH2-terminal binds DnaK accelerating

hydrolysis of ATPCOOH-terminal recognizes hydrophobic peptides

Hsp70 System

GrpE release of ADPrebinding of ATP

TRIGGER FACTOREubacteria

Binds to ribosomes Interacts with proteins > 57 residues PPIase activity Overlapping function with Hsp70

Chaperones


NACEukarya

Nascent chain-Associated Complex Lacks PPIase domain

Chaperones


Large double ring complexes ~800 kDa 2 groups

distantly related in sequencesimilar in architecture

Chaperonins

GROUP I GROUP II

EubacteriaMitochondria and chloroplast

ArchaeaEukaryotic cytosol

GroEL TRiC

GroES GroES independent

Binding and release ATP regulated

Substrate captured through hydrophobic contacts

Group I

GroEL

2 heptameric rings of 57 kDa subunits stacked back-to-back

GroES

homoheptameric ring of ~10 kDa subunits

– Equatorial domain (ATP binding site)– Hingelike domain– Apical domain

each subunit

Substrate nonnative proteins up to 60 kDa

Mayer MP.Gymnastics of Molecular Chaperones. Molecular Cell (2010) 39: 321-331

Group II

TRiC TCP-1 ring complex / CCT

8 heterogeneous subunits per ring that differ in their apical domain

Substrate: actin and tubulin

Archaeal and eukaryotic cytosol 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 themexposing hydrophobic amino acid residues

Prefoldin (PFD)

Archaea Eukarya

2 PFDα and 4 PFDβ 2 α and 4 β subunits

Binds unrelated proteins Binds actin and tubulin

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 (PFD)

Prefoldin structure

Side view of the prefoldin of M. thermautotrophicus

Bottom view of the prefoldin of M. thermautotrophicus

Prefoldin structure

50 Å 30 Å

50 Å

90 Å

85 Å70 Å

Prefoldin structure

Hydrophobicity- Bottom of the beta-barrel core- Patches near the ends of the coiled coils

PolarHydrophobic

Prefoldin structure

Hydrophobicity- Bottom of the beta-barrel core- Patches near the ends of the coiled coils

Non-PolarBasicAcidicPolar

Prefoldin structure

Prefoldin structure

Coiled coils40 residues12 turns

Beta subunitN- and C- terminal regions formα helices connected byone β hairpin linker

β hairpin 2 short β strands

Alfa subunit

Prefoldin (PFD)

2 β hairpinThe extra one for the dimerizationof α subunit monomers (residues 53-70)

Coiled coils11 and 13 turns

CATH classification:

Class: Mainly Alpha (1)

Architecture: Orthogonal Bundle (1.10)

Topology: Helix Hairpins (1.10.287)

Superfamily: (1.10.287.370)

Family: Prefoldin subunit alpha-like domainFamily: Prefoldin subunit beta-like domain

Prefoldin classification

SCOP classification:Class: All alpha proteins

Fold: Long alpha-hairpin

Superfamily: Prefoldin

Family: Prefoldin

Domain: Prefoldin alpha subunitDomain: Prefoldin beta subunit

Prefoldin classification

β

ββ

β

α

α

β-barrel Eight-stranded

Up and down

Core filled with hydrophobic amino acids

Prefoldin structure

Beta-Barrel

Prefoldin structure

Interchain interactions that stabilize the double β-barrel core of the complexMainly 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)

Prefoldin structure

Prefoldin structure

α - α

α - α

Prefoldin structure

α - β

Prefoldin structure

β - β

Prefoldin structure

α - β

Prefoldin structure

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• MtGimα2β3

• MtGimβ

• MtGimα2β2

• MtGimα2β1

• MtGimα1β2

2 MtGimα form a structural nucleus and MtGimβ subunits are organized around it

Assembly

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

a

d

Coiled coil motif 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

140 Å

22º

Prefoldin structure

22⁰

Coiled coils

Prefoldin structure

Core mean 6,290125 Å

Terminal mean 6,7275 Å

Superimposition of the α subunits of our both templates: 1FXK and 2ZDI

Archeal prefoldins

RMSD 1.43 Sc 7.73

Structural similarity

M. thermautotrophicus P. horikoshii

Superimposition of the β subunits of our both templates: 1FXK and 2ZDI

Archeal prefoldins

RMSD 1.01 Sc 8.31

Structural similarity

M. thermautotrophicus P. horikoshii

α -subunitαVal131

αVal138

αLeu14

αLeu11

N

C

Interactions with unfolded proteins

Hydrophobic grooves

β -subunits

βLeu15

βLeu12βLeu107

βLeu103

N CβVal8


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) .


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

- Cα RMSD

- 1 ns simulation time at 338K

- Force field parameter: CHARMM22

- Periodic boundary conditions


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


What we tried

Fix all the atoms except the Cαs

Increase the time of each step

Reduce the frequency of the output

Simulate at lower temperature


What the guys from the article did

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

Fluctuation of PhPFD during MD simulation

Initial modelSimulated model

What the guys from the article did


Fluctuation of PhPFD during MD simulation


PFD-Actin interaction

3D reconstruction of the complex between PFD and unfolded different substrates

Human PFD and unfolded actin

Archaeal PFD and unfolded actinArchaeal PFD

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


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

Leu111

Gly112

Ala113

Gly114

Interaction with PFDInteraction with CCTInteraction with PFD and CCT

PFD-Actin interactionUnfolded actin interacts with PFD in a quasifolded conformation that requires the chaperone protection

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


2 types of PFD-CCT interaction1. Asymmetric2. Symmetric

Interactions between the 2 chaperones occurs at the level of the PFD tentacles outer regions and the inner surface of the chaperonin apical domains

PFD oligomers interact in each ring with 2 CCT subunits placed in a 1, 4 arrangement

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 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)

Sequence Alignment

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

Secondary Structure Comparison

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

Sequence Alignment

Templates– 1FXK (from archaeal Methanothermobacter

thermautotrophicus)– 2ZDI (from archaeal Pyrococcus horikoshii)

Discarded– 3AEI & 2ZQM from Thermococcus strain KS-1

because of their incomplete structure

Modelling human PFD

Templates– 1FXK (from archaeal Methanothermobacter

thermautotrophicus)– 2ZDI (from archaeal Pyrococcus horikoshii)

Discarded–3AEI & 2ZQM from Thermococcus strain KS-1

because of their incomplete structure

Modelling human PFD

For 3AEI and 2ZQM there are no hexamers on their crystals but dimers of tetramers

Investigators were not able to crystallize the full hexamer complex and obtained two linked tetramers of β-subunits thus resembling the actual hexamer (or dimer of trimers) of PFD

Modelling human PFD

Modelling human PFD

Remember from the introduction:– Eukarya 6 different genes coding for each one of

the tentacles forming the hexamer – Archaea only two, one for α tentacles and one

for the β ones– Human subunits 3 and 5 code for α tentacles

whereas subunits 1, 2, 4 and 6 code for the β ones

Modelling human PFD

Human sequences are always longer than the ones pertaining to the available templates so it was not possible to model the entire human PFD neither with modeller or phyre

Modelling human PFD

Superimpositions for subunits 1 and 2 of both templates and the two models

β-subunits

Dark blue: modelLight blue: phyre model

Dark blue: modelGreen: phyre model

RMSD 1.87Sc 7.73

RMSD 1.94Sc 6.80

Modelling human PFD


β-subunits

Dark blue: modelLight blue: phyre model


RMSD 2.95Sc 5.95

RMSD 1.15Sc 7.97

Modelling human PFD


α-subunits

Red: modelGreen: phyre model


RMSD 1.73Sc 6.04

RMSD 2.30Sc 5.01

Modelling human PFD

Loops breaking helixes!

First problems appear: not so good alignments caused some unrealistic features

Loops breaking helixes!

Modelling human PFD

Cyan: template 1FXKGreen: template 2ZDIMagenta: modelYellow: phyre model

Subunit 1 Subunit 2

β-subunits

Check the energy profiles


Subunit 4 Subunit 6

β-subunits



Subunit 3 Subunit 5

α -subunits


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

THREADING

Highest score corresponded to 1I36 Chain A

It was meant to fail but we run modeller anyway…

THREADING

… and, obviously, it did fail.

THREADING


THREADING

The second better score was that of 2cgp, a gene activatorIt 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


THREADING

So we just run modeller again with the same template but the alignment obtained by threader

Unfolded tails again!

THREADING

Putative models of human’s prefoldin subunit 5

Threader Modeller Phyre

THREADING

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 ROSETTA in order to obtain a complete model

Check the energy profiles: conclusions

Alignment of the two human α subunits

Paralogy

Sequence similarity

Alignment of the four human β subunits

Paralogy

Sequence similarity

Escherichia coli 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 PFDNo structural homology with PFD but structural similarityCONVERGENT EVOLUTION

Curious case: Skp

Skp

Topologic diagram

PFD Skp

N C

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

RMSD 6.20

1-45

59-103

63-103

10-50

XAM

Blue: SkpRed: prefoldin


Superimposition by STAMP

RMSD 2.87Sc 2.57

Purple: SkpGreen: PFD

STAMP


Red: PFDGreen: Skp

RMSD 1.43 68 superimposed residues

RMSD 1.7852 superimposed residues


Red: SkpGreen: 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


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

PEM1) The chaperon prefoldin is present in:a) Archaeab) Eukaryac) The two previous ones are correctd) Bacteriae) 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 eubacteriab) Chaperonins need ATP for both binding and release of their substratesc) Skp is a prefoldin-like chaperone found in archaead) Chaperones mainly interact with nonnative structures by hydrophobic interactionse) Trigger Factor binds to ribosomes

3) Prefoldin has:a) Two beta subunits and four alpha subunitsb) Four beta subunits and four alpha subunitsc) Two beta hairpins and four alpha helicesd) Four beta hairpins and two alpha helicese) 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 onesb) Interactions between alpha and beta subunits are the most important onesc) The previous ones are correctd) Interactions between alpha subunits are the most importante) All of the previous answers are correct

PEM5) Select the incorrect one:a) Archaeal prefoldin binds to several nonnative structuresb) Eucaryotic prefoldin binds to actins and tubulinsc) Prefoldin has several hydrophobic residues at the terminal segments of its tentacles thus allowing its interaction with nonnative structuresd) Bacterial prefoldin needs cooperation with chaperoninse) 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 structure2. Skp and prefoldin similarities are a result of convergent evolution3. Skp chaperone is found in jellyfish, primates and humans4. N and C terminal ends of Skp chaperone are part of the trimerization core

a) 1, 2 and 3b) 1 and 3c) 2 and 4d) 4e) 1, 2, 3 and 4

7) The archaeal prefoldin interacts with: a) Actinb) Tubulin c) The previous ones are correctd) 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 regionsc) Exclusively hydrophilic regionsd) All the answers are wronge) 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 flexibilityb) To study the flexibility of the tentacles is necessary a molecular dynamics simulationc) The previous ones are correctd) Tentacles are the most flexible part of the prefoldin chaperonee) All the previous statements are correct

10) What is true about the coiled-coil motif:f) It is a motif consisting of alpha-helices and beta-strandsg) It is characterized by a heptad repeat in which the first (a) and fourth (d) residues tend to be hydrophobich) Both previous statements are correcti) The hydrophobicity of the coiled-coil core helps to achieve the periodicity of the heptad repeatj) All previous statements are correct.

Prefoldin structure

β - β

T-coffee: What is the meaning of the color coded html output?

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:

To understand the formula...

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.

Extended scores (ES)

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.

Skp

SCOP classification:Class: Membrane and cell surface proteins and peptides

Fold: OmpH-likeSuperfamily: OmpH-like

Family: OmpH-likeDomain: Periplasmic chaperon skp (HlpA)

Species: Escherichia coli [TaxId: 562]

CATH classification:Class: Alpha Beta

Architecture: 2-Layer SandwichTopology: Protein Binding, DinI Protein; Chain A

Homology: OmpH-like (Pfam 03938)

Roger Bartomeus Jolita Jancyte Helena Palma Anna Perlas.

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