Accurate Structural Modelling of the Interaction of a Designed Ankyrin Repeat Protein with the Human...

Accurate Structural Modelling of the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2CSIRO Materials Science & Engineering, Parkville, Vic. 3052.

Vidana C. Epa ([email protected])22 March 2013

CMSE

Human Epidermal Growth Factor Receptor 2 (HER2) & Designed Ankyrin Repeat Protein G3

• HER2, a cell-surface receptor with an intracellular Tyrosine kinase domain and an ectodomain (~600 a.a.) with 4 distinct structural domains.

• Human epidermal growth factor receptor 2 (HER2, ErbB2) over-expressed in ~30% of breast tumors and indicative of poor prognosis.

• HER2 an important target for cancer therapeutic and diagnostic development.

• Of the 2 anti-HER2 mAbs in the clinic, trastuzumab (Herceptin) binds to domain 4 and pertuzumab (Perjeta) binds to domain 2.

• Designed ankyrin repeat proteins (DARPins) are a novel class of small highly stable proteins that can be selected by ribosome display to bind target proteins with high affinity.

• The DARPin H10-2-G3 (“G3”) has been selected to bind HER2 with picomolar affinity.

← domain 4

How does the DARPin G3 interact with HER2?• HER2, a cell-surface receptor with an intracellular

Tyrosine kinase domain and an ectodomain (~600 a.a.) with 4 distinct structural domains.

• Human epidermal growth factor receptor 2 (HER2, ERbB2) over-expressed in ~30% of breast tumors and indicative of poor prognosis.

• HER2 an important target for cancer therapeutic and diagnostic development.

• Of the 2 anti-HER2 mAbs in the clinic, trastuzumab (Herceptin) binds to domain 4 and pertuzumab (Perjeta) binds to domain 2.

• Designed ankyrin repeat proteins (DARPins) are a novel class of small highly stable proteins that can be selected by ribosome display to bind target proteins with high affinity.

• The DARPin H10-2-G3 (“G3”) has been selected to bind HER2 with picomolar affinity.

← domain 4

TCSS-FOAM 2013: Protein-Protein Interactions | Vidana Epa

Macromolecular Docking - Rigid-body docking of 2 protein structures

•ZDOCK ( Zhiping Weng, Boston U.)•Grid-based rigid body docking algorithm (originally from E. Katchalski-Katzir)•Discretizes the two proteins ‘receptor’ and ‘ligand’ into grids, and performs a global scan of the rotational and translational space of the ligand with respect to the receptor.•Each relative orientation scored by a simple pairwise shape complementarity (PSC) scoring function.•For every rotation, translational space is rapidly scanned using Fast Fourier Transforms. 2000 docked solutions.

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Grid spacing: 1.2 ÅRotational sampling: 60


Rigid-body docking of 2 protein structures

•ZDOCK ( Zhiping Weng, Boston U.)•Grid-based rigid body docking algorithm (originally from E. Katchalski-Katzir)•Discretizes the two proteins ‘receptor’ and ‘ligand’ into grids, and performs a global scan of the rotational and translational space of the ligand with respect to the receptor.•Each relative orientation scored by a simple pairwise shape complementarity (PSC) scoring function.•For every rotation, translational space is rapidly scanned using Fast Fourier Transforms.•Initial-stage rigid body docking solutions (2000) are next re-ranked with an optimized energy function, ZRANK.•ZRANK function is a linear weighted sum of van der Waals, Coulomb, and desolvation energy terms.

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Using existing experimental data to guide the docking (filtering)

• No evidence that G3 binds to domains 1-3 of HER2. Hence use only domain 4 for the docking.

• Concave face of DARPins used in binding to target proteins. Hence ‘block out’ docking solutions that involve HER2 interacting with the convex face of G3.


ZRANKed docking solutions from ZDOCK

ZRANK # ZDOCK solution #1 16982 13 5664 455 4316 207 10948 419 38010 13611 24012 8813 2114 2415 123516 26817 618 19519 49120 8

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Metric #1: Total Interaction Energy (IE)

• When 2 proteins form a complex, favourable interactions between the two are maximized to give the most stability.

• Compute the total interaction energy (IE) between the 2 proteins.• Use the empirical force-field FoldX (J. Schymowitz et al., 2005) to calculate IE.• IE contains a linear combination of van der Waals energy, solvation, hydrogen

bonding, Coulomb electrostatics, and entropy change terms.

Metric #2: Shape Complementarity (Sc)

• When 2 proteins form a complex, favourable interactions between the two are maximized to give the most stability.

• Compute the total interaction energy (IE) between the 2 proteins.• Use the empirical force-field FoldX (J. Schymowitz et al., 2005) to calculate IE.• IE contains a linear combination of van der Waals energy, solvation, hydrogen

bonding, Coulomb electrostatics, and entropy change terms.

• In protein-protein interactions, the interaction is maximized largely by maximizing the shape complementarity at the interface, leading to maximizing favourable van der Waals interactions between the 2 proteins.

• Compute the shape complementarity (Sc) between the 2 proteins at the interface. (Lawrence & Colman, 1993).

• Sc=1.0 for prefect complementarity.


ZRANKed solutions scored with metrics Sc and IE

ZRANK # ZDOCK solution # Sc IE (kcal mol-1)1 1698 0.425 -6.352 1 0.704 -15.403 566 0.663 -8.514 45 0.748 -19.375 431 0.668 -3.616 20 0.673 -14.297 1094 0.414 -3.728 41 0.672 -13.469 380 0.658 -10.4210 136 0.710 -13.3311 240 0.400 -0.9912 88 0.444 -10.9813 21 0.520 -15.4214 24 0.557 -13.6515 1235 0.492 -8.2916 268 0.386 -3.1917 6 0.610 -15.7518 195 0.550 -18.6619 491 0.365 -6.1520 8 0.529 -12.72

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Top solution selected (#45)


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Visual examination of solutions assists in the elimination process


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ZDOCK solutions #1, 136 result in probable clashes with the cell membrane.


Yet another metric, ECZRANK # ZDOCK solution # Sc IE (kcal mol-1)1 1698 0.425 -6.352 1 0.704 -15.403 566 0.663 -8.514 45 0.748 -19.375 431 0.668 -3.616 20 0.673 -14.297 1094 0.414 -3.728 41 0.672 -13.469 380 0.658 -10.4210 136 0.710 -13.3311 240 0.400 -0.9912 88 0.444 -10.9813 21 0.520 -15.4214 24 0.557 -13.6515 1235 0.492 -8.2916 268 0.386 -3.1917 6 0.610 -15.7518 195 0.550 -18.6619 491 0.365 -6.1520 8 0.529 -12.72

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Electrostatic Complementarity at Protein-Protein Interfaces, EC (McCoy, Epa & Colman, JMB, 268, 270(1997))

• Calculate the correlation coefficients between the electrostatic potentials (from solving the linearized Poisson-Boltzmann equation) at the buried molecular surface of one protein due to the partial charges on the other protein and itself.

• Metric #3: EC Confirms that solution #45 is the preferred one.

Electrostatic potentials generated by N9 Neuraminidase and NC41 and NC10 Fabs on the buried molecular surface of the complex.

(blue): (+)ve e.s. potential; (red): (-)ve e.s. potential


Computational model of the structure of the complex between HER2 and G3 (after energy refinement of solution #45)

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TCSS-FOAM 2013: Protein-Protein Interactions| Vidana Epa

Herceptin superimposed on the HER2-G3 complex model

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Interactions between HER2 and G3

•Majority of the interactions at the HER2 (blue) – G3 (red) interface are of van der Waals in nature.•Relatively large number of the G3 interacting residues are aromatic.•Exposed Tyr residues well-known for facilitating protein-protein interactions.•Majority of the interacting amino acid residues on G3 are at randomized positions.

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Experimental Validation of the computational structural model

• Mutate HER2 amino acid residues at the interface predicted by the model to make important interactions and stabilize the complex.

• Selected HER2 residues Leu 525, Ser 551, Val 552, and Phe 555.

• Alanine mutants of these residues should decrease binding to G3, not destabilize HER2 itself, and have no effect on the binding of Herceptin.

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Surface Plasmon Resonance (SPR) results for the interaction of HER2 and mutants with G3 and Herceptin

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HER2 Mutation KD,MUTANT / KD,WILDTYPE for G3 binding

KD,MUTANT / KD,WILDTYPE for Herceptin binding

Wild-type 1.0 1.0

Leu525-Ala 79.7 1.0

Ser551-Ala 3.8 1.0

Val552-Ala 114.4 1.0

Phe555-Ala 63.2 1.1


Comparison of the computational model with the X-ray crystal structure

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Backbone RMSD: 0.84 Å, 1.14 Å

RMSD for interface residues: 0.92 Å

24 out of 26 residue interactions across the interface are present in the model.→ fnat=0.92

→ This model can be classed as “highly accurate” according to CAPRI assessment criteria.


Acknowledgements

• Tim Adams (CMSE)• Xiaowen Xiao (CMSE)• Olan Dolezal (CMSE)• Larissa Doughty (CMSE)• Andreas Plückthun (Univ. of Zurich)• Christian Jost (Univ. of Zurich)

• V.C. Epa et al., PLoS One, 8(3), e59163 (March 2013).

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Accurate Structural Modelling of the Interaction of a Designed Ankyrin Repeat Protein with the Human...

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