Folding, Assembly, Flexible Systems Maxim Petoukhov EMBL, Hamburg - - PowerPoint PPT Presentation

Folding, Assembly, Flexible Systems

Maxim Petoukhov EMBL, Hamburg Outstation

• Introduction
• Combined rigid body and ab initio modelling
• Unfolded proteins
• Modular proteins with disordered linkers
• Transient complexes and weak oligomers
• Examples
• Conclusions

Outline Outline

• Conventional rigid body modelling allows

quaternary structure analysis of macromolecular complexes, oligomers and modular proteins

• The use of complementary data significantly

reduces the ambiguity

– The resulting models are still of low resolution

Re: Atomic Structure Based Modelling Re: Atomic Structure Based Modelling

∑

=

k k k

s I v s I ) ( ) (

Re: Quantitative Analysis of Mixtures

OLIGOMER: volume fractions of individual components Prerequisite: models (profiles) of components are known

s, nm-1 5 10 15 lg I(s) 5 6 7 8 Resolution, nm 2.00 1.00 0.67 0.50 0.33

Small angle scattering: resolution

Shape F

• ld

Atomic structure

Resolution is defined by the magnitude of the largest scattering vector in the diffraction pattern d=2π/smax

Concept of Dummy Residues Concept of Dummy Residues

• Proteins are (folded) polypeptide chains composed of

amino acids

• At a resolution of ~1 nm each amino acid can be

represented as one entity (dummy residue)

• For simplicity DRs are

– Identical – Centered at the Cα positions

= < … … >

D.I. Svergun, M.V. Petoukhov, & M.H.J. Koch (2001) Biophys. J. 80, 2946-53

Building native-like folds of polypeptides

Primary sequence Secondary structure Excluded volume

Neighbors distribution Knowledge-based potentials Bond angles & dihedrals distribution

• Using DR-type models and protein-specific penalty functions

(Mis)-Folding from SAXS?

• “Native-like” folds of lysozyme

Modelling Modelling of

• f multidomain

multidomain proteins proteins

MRGSHHHHHH GSGVPSRVIH IRKLPIDVTE GEVISLGLPF GKVTNLLMLK GKNQAFIEMN TEEAANTMVN YYTSVTPVLR GQPIYIQFSN HKELKTDSSP NQARAQAALQ AVNSVQSGNL ALAASAAAVD AGMAMAGQSP VLRIIVENLF YPVTLDVLHQ IFSKFGTVLK IITFTKNNQF QALLQYADPV SAQHAKLSLD GQNIYNACCT LRIDFSKLTS LNVKYNNDKS RDYTRPDLPS GDSQPSLDQT MAAAFGLSVP NVHGALAPLA IPSAAAAAAA AGRIAIPGLA GAGNSVLLVS NLNPERVTPQ SLFILFGVYG DVQRVKILFN KKENALVQMA DGNQAQLAMS HLNGHKLHGK PIRITLSKHQ NVQLPREGQE DQGLTKDYGN SPLHRFKKPG

Modelling Modelling of

• f multidomain

multidomain proteins proteins

• A

combined approach is proposed to built the models

• f multidomain proteins with

large and flexible interdomain linkers

• The latter are represented as

DR chains which are attached to the appropriate terminals in rigid domains.

• A single modification of a

model is a rotation about one

• r two randomly selected

DR(s).

Modelling Modelling of

• f multidomain

multidomain proteins proteins

Building native Building native-

• like folds of linkers

like folds of linkers

Absence of steric clashes

• 150
• 100
• 50

Bond angles & dihedrals distribution Loop compactness may also be required

3

3

l id

n Rg =

Neighbors distribution along the sequence

i i+K r

s, nm-1

0.5 1.0 1.5 2.0

lg I, relative

8 9 10 11

Simultaneous fitting of multiple data sets from Simultaneous fitting of multiple data sets from deletion mutants deletion mutants

BUNCH: BUNCH: Modelling Modelling of

• f multidomain

multidomain proteins proteins

• Search of the optimal positions and orientations of rigid domains

and probable conformations of DR linkers, those fit the SAXS data.

• Proper bond and dihedral angles in the DR chains are required

together with the absence of overlaps.

• The scattering pattern is calculated from partial amplitudes of

domains and form-factors of DR comprising the loops using spherical harmonics.

• Multiple scattering curves fitting from deletion mutants

( ) ( ) ( ) 2

) ( ) ( 2

| | 2

∑ ∑ ∑ ∑

+ =

∞ = − = i lm i l l l m k lm k

s D s A s I π

Petoukhov M.V., Svergun, D.I. (2005). Biophys. J. 89, 1237-1250

Structure and RNA interactions of polypyrimidine tract binding protein

NMR: high resolution structures

• f RRM1 and RRM2

Collaboration: S.Curry (London)

A B C D

Multiple scattering curves from deletion mutants fitted simultaneously

PTB is an important regulator of alternative splicing, which allows the production of multiple mRNA transcripts from a single pre-mRNA species. PTB contains four domains (RNA recognition motifs, RRMs), whose structure is solved by NMR.

Structure and RNA interactions of polypyrimidine tract binding protein

Overlap of the typical ab initio and rigid body models Further restraints (e.g. from NMR) are required to resolve the orientational ambiguity

Petoukhov, M. V., Monie, T. P., Allain, F. H., Matthews, S., Curry, S., and Svergun, D. I. (2006). Structure 14, 1021-1027.

CORAL: Crossing SASREF & BUNCH

• Sasref:

– Does not account for missing portions

• Bunch:

– Single polypeptide chain – Impossible to fix more than one domain

Random Loop Library for Combined Modelling

RANLOGS database

CORAL

Hybrid Modelling in Coral

RANLOGS database

CORAL

Novel feature: consorted movements

Kratky Plots to Detect Disorder

Unfolded Folded Multi-domains with flexible linkers

Patterns of globular and flexible proteins

► Local Protein Fluctuations: Backbone and side-chains Protein vibrations, loop motions and breathing to facilitate interactions and catalysis ► Concerted domain motions: Linkers as Hinges Specific and limited domain jumps between relative positions often linked with catalysis in on/off mechanisms ► Highly flexible regions or domains An astronomical number of conformations are available. Flexible multi-domain proteins (MD) and Intrinsically Disordered Proteins (IDPs) Linked with signaling and regulation

Biomolecules are Dynamic Entities

Flexible Proteins: Importance

► Many biological functions such as transcription, regulation, cell cycle control, require extensive flexibility ► Is more common in higher organisms that have to perform more and more controlled functions. Disorder is correlated with complexity ► High selectivity, moderate affinity, and promiscuity are properties often linked to flexibility ► In these systems partially structured conformations or transient long range interactions can be crucial for biological

• activity. Structural studies are important

► Smooth Scattering profiles and featureless Kratky Plots ► Large Rg and Dmax ► Absence of correlation peaks in the p(r) function ► Low correlation densities in ab initio reconstructions ► Isolated domains in rigid body modelling ► Prediction of disorder using bioinformatics tools

http://www.idpbynmr.eu/home/science/research-tools.html

Indications (not Proofs!) of Flexibility

Flexibility as mix of different conformations

∑

=

k k k

s I v s I ) ( ) (

dr sr sr r p s I

D

∫

= sin ) ( 4 ) ( π

vk = volume fraction Ik(s) = scattering intensity from the k-th component For monodisperse systems the scattering is proportional to that of a single particle averaged

• ver all orientations

Detection of Flexibility

PolyUbiquitin Molecules

2,3,4 and 5 Ubiquitin (72 AA) domains connected by 20 AA linker (RanCH)

Flexible Rigid

Flexible Multidomain Proteins present less features than rigid counterparts Bernadó Eur. Biophys. J. 2009, 39, 769

SAXS curves

Analysis of the overall size descriptors (Rg, p(r), Kratky)

Modelling: ab initio (DAMMIN/DAMMIF) and Rigid body (BUNCH/CORAL) Analysis of the differences

Rigid Scenario Flexible Scenario

Detection of Flexibility

Go for flexibility!

Ensemble Optimization Method

structures curve

Ensemble Optimization Method

...

Rg

1

Rg

2

Rg

3

Rg

4

Rg

5 ...

ρ (Rg)

...

∑

=

I N s I

) ( 1 ) (

Ensemble methods in SAXS

Elitism

Experimental Curve

Mutations C r

• s

s i n g N

Rg Distribution

Elitism

Experimental Curve

Mutations Crossing N G Generations

…

R1 R2 R3 R2 R1 R3

…

Ensemble Optimization Method (EOM)

Bernadó, Mylonas, Petoukhov, Blackledge, and Svergun. J Am Chem Soc 2007, 129:5656-64.

ψ φ

Cα Cα

Kohn et al. PNAS, 2004, 101, 12491 Rg

Rg = R0·Nν R0 Persistence Length ν Solvent ‘quality’

Several experimental and theoretical studies establish ν ≈ 0.588 as an indication of the ‘random coil’ in chemically denatured (Urea or GuHCl) proteins. N

Theoretical distribution of the bond and dihedral angles for random chains Quasi Cα -Cα Ramachandran plot

• G. Kleywegt , Validation of protein models from Cα

coordinates alone, JMB, 1997, 273, 371-376

Bond angles vs. Dihedral angles

Modelling: Native vs. Random

Missing loops (i.e. flat electron density

map) …

MRIGMV……..GGVQSHVLQ…..VLRDAGHEVS…….PHVKLPDYVS

missing loop 30 AA

Kratky Plot

apoferritin

vs.

pool

Nter.pdb Cter.pdb seq.seq curve.dat

Rg, Å Dmax, Å

Multi-curves fitting

pool

EOM 2.0 can handle multimeric assemblies

(full length protein measured in two buffers with low and high ionic strength respectively)

EOM: Impact of the Pool Size

… the beauty of the Pentagon

• G. Tria

Ensemble Optimization Method

curve actual structures Optimized ensemble

Assessing conformation variability: Assessing conformation variability: lysozyme lysozyme u unfolding nfolding

8M Urea, 10 mM DTT 8M Urea, 10 mM DTT Rg = 26.3 Å Rg (native) = 15.1 Å 8M Urea, 100 mM DTT Rg = 30.0 Å

• E. Mylonas

…

FULL-LENGTH

Pool

M CONFORMATIONS (POOL)

…

DELETION-1

Pool-1

…

DELETION-2

Pool-2

EOM-1

…=

EOM

…= …=

EOM-2 Cα-Cα Average Distance Matrix

Experimental data

Multiple Curve Fitting with EOM: Reaching «Higher Resolution»

Adult Tau Fetal Tau

R1R2R3R4 P2 P1 I2 I1 N C R1R2R3R4 P2 R1R2R3R4 P2 P1 R1R2R3R4

K32 K16 K18 ht40

R1 R3R4 P2 P1 N C R1 R3R4 P2 R1 R3R4 P2

K27 K17 K19 K44

R4 R1 R3 R1 R3R4 P2 P1 N R1 R3R4 C

K10

P2 P1 N

K25 ht23

Mylonas et al. Biochemistry 2008, 47, 10345 <Cα-Cα>select <Cα-Cα>pool <Cα-Cα>select <Cα-Cα>pool

Application to Tau Protein

Other ensemble approaches

Maximum Occurrence Approach

• The MO of a conformation can be

calculated as the maximum weight that the conformation can have and be in agreement with the experimental data

– NMR data » Residual dipolar couplings » Pseudo contact shifts » SAXS

Paramagnetic Me

Bertini, Giachetti, Luchinat, Parigi, Petoukhov, Pierattelli, Ravera, Svergun, JACS, 2010

Maximum Occurrence Approach

Probe of Calmodulin Conformations by Combining SAXS with NMR

Blue: 5% Red: 40%

• E. coli
• E. coli Flavorubredoxin

Flavorubredoxin

Modular enzyme endowed with nitric oxide and/or oxygen reductase activity

Collaboration:

• J. Vicente and P.Crowley (ITQB, Lisboa)

?

• E. coli
• E. coli Flavorubredoxin

Flavorubredoxin: : ab ab initio initio modelling modelling

• E. coli
• E. coli Flavorubredoxin

Flavorubredoxin: : Xtal Xtal vs vs SAXS SAXS

• E. coli
• E. coli Flavorubredoxin

Flavorubredoxin: : rigid body modelling rigid body modelling

• E. coli
• E. coli Flavorubredoxin

Flavorubredoxin: various : various constraints constraints

• E. coli
• E. coli Flavorubredoxin

Flavorubredoxin: EOM : EOM

Heterogeneous Assemblies with Flexibility

Cross-shaped extended p53 from SAXS and NMR

Tumor suppressor p53 and its complex with DNA

P53 is a transcription factor that regulates genes involved in cell cycle and apoptosis. Misfunction of p53 is related with cancer The homotetrameric p53 consists of folded core and tetramerization domains, linked and flanked by intrinsically disordered segments Tidow, H., Melero, R., Mylonas, E., Freund, S.M., Grossmann, J.G., Carazo, J.M., Svergun, D.I., Valle, M. & Fersht, A.R. (2007) Proc Natl Acad Sci USA, 104, 12324

Compact P53/DNA from SAXS also confirmed by EM

NC-dUTPase interactions with oligonucleotide

Németh-Pongrácz, V., Barabás, O., Fuxreiter, M., Simon, I., Pichová, I., Rumlová, M., Zábranská, H., Svergun, D., Petoukhov, M., Harmat, V., Klement, É., Hunyadi-Gulyás, É., Medzihradszky, K., Kónya, E. & Vértessy B. (2007) Nucleic Acid Res. 35, 495-505.

The homotrimeric fusion protein nucleocapsid (NC)- dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. + nucleocapsid dUtpase (TG)4 Xtal NMR

(1) NC-dUtpase (2) NC-dUtpase+peptide (3) NC (4) NC+peptide

P1 P3

• Compactisation of loaded NC
• No contacts between the core and NC
• Flexibility
• Quasi-P3 symmetry may persist

Complex Complex Stoichiometry Stoichiometry

R.H.H. van den Heuvel, D.I. Svergun, M.V. Petoukhov, A. Coda, B. Curti, S. Ravasio, M.A. Vanoni & A. Mattevi (2003). J. Mol. Biol. 330, 113-128

+

s, nm-1

1 2 3

• 3
• 2
• 1

1 2 3 4

lg I(s), relative

Fd Fd-Glts Fd:Fd-Glts 1:1 Fd:Fd-Glts 2:1

Equimolar mixture

• f Fd:Fd-Glts 1:1

and free Fd

Molecular Assembly of Lumazine Synthase

s, nm-1

0.2 0.4 0.6 0.8 1.0 1.2 1.4

lgI, relative

• 1

1 2 3 4 5 Experiment Icosahedral P1 pH 7 pH 10

r, nm

5 10 15 20 25 30

p(r), relative

0.2 0.4 0.6 0.8 1.0

pH 7 pH10

2nm

pH 7 pH 10 LS catalyzes the formation of 6,7-dimethyl-8-ribityllumazine in the penultimate step

• f riboflavin biosynthesis. Depending on the buffer it forms pentamers, dimers of

pentamers and icosahedral capsids Wild type LS in borate buffer T1 T4 (?)

Zhang, X., Konarev, P.V., Petoukhov, M.V., Svergun, D.I., Xing, L., Cheng, R.H., Haase, I., Fischer, M., Bacher, A., Ladenstein, R. & Meining, W. (2006) J Mol Biol. 362, 753-770

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

• Timely inhibition of Cdt1 by Geminin is essential to this DNA replication licensing
• The mechanism of DNA licensing inhibition by Geminin, is analyzed by combining

MX, SAXS and functional studies.

• The Cdt1:Geminin complex can exist in two distinct forms, a ‘‘permissive’’

heterotrimer and an ‘‘inhibitory’’ heterohexamer

• V. De Marco, P. J. Gillespie, A. Lib, N. Karantzelis, E. Christodouloua, R. Klompmaker, S. van Gerwen, A. Fish,
• M. V. Petoukhov, M. S. Iliou, Z. Lygerou, R. H. Medema, J. J. Blow, D. I. Svergun, S. Taraviras & A. Perrakis

(2009) PNAS USA, 106, 19807

s, nm-1

0.5 1.0 1.5 2.0

lg I, relative

2 3 4

Calmodulin-Activated Glutamate Decarboxylase

Gut H, Dominici P, Pilati S, Astegno A, Petoukhov MV, Svergun DI, Grütter MG and Capitani G. J Mol Biol. 2009 392:334-51

• 6:3 Gad:CaM stoichiometry
• Planar arrangement of CaM
• In proximity of the three 2-fold

axes of Gad

GASBOR_MX: quaternary structure of weak (symmetric)

• ligomers

SASREF_MX: structural analysis of transient complexes

3D Reconstruction from Polydisperse Data?

BSA Example

• Untreated sample
• Purified sample
• Fit by 3v03

BSA Example

SASREF_MX GASBOR_MX

• SAS van address protein flexibility, conformational

changes and assembly/dissociation processes

• Ensemble Methods are appropriate tools to study

(potentially) flexible molecules

• Unique structural information can be obtained based
• n distributions of descriptors whereas structures

collected are simply a TOOL to describe the shape distributions

• Transient complexes and weak oligomers may be

modelled (with caution) against SAXS data

• Protein fold can not (yet?) be obtained from SAS
• Hybrid modelling is applied for modular proteins with

moderate flexibility

Summary Summary