Roger Bartomeus Jolita Jancyte Helena Palma Anna Perlas.
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Transcript of 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
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein. Science 295 (2002): 1852-58
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
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein. Science 295 (2002): 1852-58
NACEukarya
Nascent chain-Associated Complex Lacks PPIase domain
Chaperones
Hartl FU, Hayer-Hartl M. Molecular Chaperones in the Cytosol: from Nascent Chain to Folded Protein. Science 295 (2002): 1852-58
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
α - β
Prefoldin structure
β - β
Prefoldin structure
α - 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
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) .
Interactions with unfolded proteins
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
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
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 Cαs
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
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
Flexibility of the tentacles
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
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-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
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
PFD-Actin interaction
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
Superimpositions for subunits 4 and 6 of both templates and the two models
β-subunits
Dark blue: modelLight blue: phyre model
Dark blue: modelGreen: phyre model
RMSD 2.95Sc 5.95
RMSD 1.15Sc 7.97
Modelling human PFD
Superimpositions for subunits 3 and 5 of both templates and the two models
α-subunits
Red: modelGreen: phyre model
Dark blue: 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
Cyan: template 1FXKGreen: template 2ZDIMagenta: modelYellow: phyre model
Subunit 4 Subunit 6
β-subunits
Check the energy profiles
Cyan: template 1FXKGreen: template 2ZDIMagenta: modelYellow: phyre model
Subunit 3 Subunit 5
α -subunits
Check the energy profiles
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
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 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
Higher scores of THREADER for the human subunit 5 of prefoldin (an α subunit)
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 of Skp and PFD
Superimposition by STAMP
RMSD 2.87Sc 2.57
Purple: SkpGreen: PFD
STAMP
Superimposition of Skp and PFD
Superimposition of Skp and PFD
Red: PFDGreen: Skp
RMSD 1.43 68 superimposed residues
RMSD 1.7852 superimposed residues
Superimposition of Skp and PFD
Red: SkpGreen: 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
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)