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Università degli Studi di Milano Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”. Protein modeling by fragmental approach: connecting global homologies with local peculiarities. Alessandro Pedretti. Molecular docking. Molecular dynamics. Protein modelling. Structure-based studies.

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Università degli Studi di Milano

Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”

Protein modeling by fragmental approach:

connecting global homologies

with local peculiarities

Alessandro Pedretti


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Molecular docking

Molecular dynamics

Protein modelling

Structure-based studies

  • In order to perform structure-based studies as:

    • ligand optimization;

    • virtual screening;

    • signal transduction;

    • substrate recognition.

  • the 3D structure of the biological target is required.

  • Unluckily, the experimental structure (X-ray diffraction or NMR) is not available for all proteins.


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GFGPHQRLEKLDSLLS…

1D structure

Protein modelling

3D structure

Comparative modelling

Protein modelling

Ab-initio modelling

What’s the protein modelling ?

  • The protein modelling allows to obtain the 3D structure of a protein from its aminoacid sequence (primary structure):

  • It can be classified into two main approaches:


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Structures obtained by experimental approaches (X-ray and NMR).

Comparative modelling

  • It’s based on the assumption: proteins with high homology of sequence should have similar folding.

Target sequence

3D structure database

3D template

Homology > 70 %

Alignment

Between target and template

Rough 3D model

To refinement workflow


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Ab-initio NMR). modelling

  • It’s based on physical principles and geometric rules obtained by sequence and structure analysis of the 3D experimental models.

Target sequence

Folding prediction

Application of physical and geometric rules

Multiple solutions

Global optimization

By MM and stochastic approaches

Rough 3D model

To refinement workflow


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Comparative vs. ab-initio NMR). modelling

*Models that are structurally similar due to the common template.

  • The possibility to obtain structural “clones” is very high, submitting whole query sequences of protein families with high homology to a limited number of 3D templates (e.g. transmembrane proteins).


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Fragmental approach NMR).

Target sequence

Fragmentation in structural domains

Done on the basis of information included in

databases and/or domain finder tools.

Folding prediction of each fragment

Trough multiple comparative modelling procedures.

By geometric superimposition with the 3D structure of the global template, using molecular modelling tools as VEGA ZZ.

Assembling using the global 3D template

Rough model

To refinement workflow


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Model refinement procedure NMR).

Rough model

VEGA ZZ

+

NAMD

Missing residues

Side chains add

Hydrogens add

Energy minimization

Structure check

Final model


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17 subunit types NMR).

a1, b1, , d,e

a2-10, b2-4

  • The complete model didn’t exist.

  • The design of selective a4b2 ligands is problematic due to the low information about the binding mode.

Human

a4b2 subtype

Muscle

Nervous system

Human a4b2 nicotinic receptor

  • The nicotinic acetylcholine receptors (nAchRs) are composed by five subunits assembled around a central pore permeable to cations.

  • The therapeutic interest on nicotinic ligands is highlighted by diseases involving the nAchRs as: Alzheimer’s and Parkinson’s disease, autism, epilepsy, schizophrenia, depression, etc.

Pedretti A. et Al., Biochemical and Biophysical Research Communications, Vol. 369, 648–53 (2008).


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4 transmembrane domains NMR).

2 cytoplasmic loops

1 extracellular loop

2 terminal domains

Fragmentation

Primary structure

MM refinement

Final monomer

Monomer modeling

SwissProt

Folding prediction of each fragment

Fugue

The docking results were filtered the Torpedo Californica nAChR structure.

Helices assembly by molecular docking

ESCHER NG

Full assembly

Side chains

VEGA ZZ

Hydrogens

VEGA ZZ + NAMD


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Side view NMR).

Top view

Complex assembling

2x a4

Multistep docking:

a4 + b2 → a4b2

2 a4b2 → (a4)2(b2)2

b2 + (a4)2(b2)2 → (a4)2(b2)3

+

a4b2

ESCHER NG

3x b2


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Nicotine NMR).

Epibatidine

ABT-418

Citisine

A-85380

VEGA ZZ

FRED 2

NAMD

Ligand

Binding site selection

Trp182, Cys225, Cys226 in a4

+

Docking

Minimization

a4b2 receptor

Final complex

Model validation

  • The soundness of the resulting model was checked docking a set of know nicotinic ligands:

  • All these ligands were simulated in their ionized form.


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Trp82 NMR).b2

Cys225 a4

Asn134 b2

Cys226 a4

Phe144 b2

Trp182 a4

Docking results

  • After the final MM minimization, the docking scores were recalculated by Fred 2 (ChemGauss2 scoring function):

a4b2 – nicotine complex


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Metabotropic receptor NMR).

Ionotropic receptor

Human glutamate transporter EAAT1

  • L-glutamate is the main excitatory neurotransmitter in the CNS.

Synaptic cleft

Axon

Dendrite

Excitatory effects

Glutamate

EAAT1-5

  • It can also overactivate the ionotropic receptors, inducing a series of destructive processes involved in acute and chronic neurological diseases (e.g. amyotrophic lateral sclerosis, Alzheimer’s disease, epilepsy, CNS ischemia, etc).

Pedretti A. et Al., ChemMedChem, Vol. 3, 79-90 (2008).


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EAAT ligand classification NMR).

  • They can be classified in:

  • Natural substrates.

  • Substrate inhibitors.

  • Non transported uptake blockers.

  • The last two classes are interesting because in pathological conditions, when the electrochemical gradient is damaged, EAATs can operate in reverse mode, overactivating the post-synaptic receptors.

  • Research aims:

  • Human EAAT-1 3D structure by homology modeling.

  • Pharmacophore models for all ligand classes.


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Fragmentation NMR).

Primary structure

Folding prediction of each fragment

Fugue

The assembly was carried out using the crystal structure of glutamate transporter homologue from Pyrococcus horikoshii.

MM refinement

Final monomer

VEGA ZZ + NAMD

Monomer modeling

The domains were found aligning the sequences of EAAT1 and glutamate transporter from Pyrococcus horikoshii.

SwissProt

Full assembly

Hydrogens

VEGA ZZ

Side chains


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Complex assembling NMR).

ESCHER NG

VEGA ZZ + NAMD

Monomer

Homotrimer

  • Complex refinement protocol:

  • 1 ns of simulation time;

  • restrained transmembrane segments;

  • final conjugate gradients minimization.

DEEP surface


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Docking NMR).

Mopac 7

FlexX

Ligand

Minimization

Complex

EAAT1 monomer

Docking studies

  • Two ligand subsets were docked:

    • natural substrates and competitive substrates inhibitors (16);

    • non-transported blockers (16).

  • The following procedure was applied to all ligands:

  • The docking analyses were focused on residues enclosed in a sphere centered on Arg479 (TM4). Mutagenesis studies showed this residue plays a pivotal role in the substrate interaction.


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Docking results: substrate inhibitors NMR).

Met451

Val449

Arg479

Thr450

Gln204

EAAT1 – (2S, 4R)-methylglutamate complex

Gln445

pKm = 4.88 (±0.04) – 1.52 (±0.12) Vover

N = 15, r2 = 0.93, s = 0.11, F = 174.11

Where Vover is maximum overlapping volume between the ligand and EAAT1 computed by FlexX.


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Leu448 NMR).

Ile465

Val449

Ile468

Thr450

Trp473

Arg479

Gln445

Gln204

Docking results: non-transported blockers

EAAT1 – L-TBOA complex

pIC50 = 0.4446(±0.07) – 0.141(±0.02)ScoreFlexX

N = 16, r2 = 0.77, s = 0.55, F = 43.46


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Natural and substrate inhibitors NMR).

Non-transported blockers

En = excluded volume

An = H-bond acceptors

P = ionisable group (positively charged)

Y = hydrophobic region

L-glutamate

TFB-TBOA

Pharmacophore mapping

  • The two pharmacophore models were obtained by Catalyst 4 software.

  • Both models highlight the key features required for the interaction.

  • Mapping the docking results onto the pharmacophores, it’s possible to highlight the two approaches are successfully overlapped.


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Conclusions NMR).

  • We obtained the full model of two transmembrane protein through the fragmental approach.

  • Performing molecular docking studies, we highlighted the main interaction between ligands and the proteins that were confirmed by experimental data, obtained by mutagenesis studies.

  • Although the number of considered ligands isn’t statistically relevant, we obtained good relationships between the docking scores and the experimental data, confirming the soundness of both models.

  • All these results show the power and the goodness of the fragmental approach that is able to overcame the problems introduced by global homologies and the possibility to obtain structural clones.


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www.vegazz.net NMR).

www.ddl.unimi.it

Acknowledgments

  • Giulio Vistoli

  • Cristina Marconi

  • Cristina Sciarrillo

  • Laura De Luca


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