• Integrins are a large family of αβ heterodimeric cell surface receptors found in multicellular organisms.

• Integrin αvβ6 heterodimer is an epithelial-restricted transmembrane glycoprotein.

• Expressed at low-levels in normal cells, elevated in wound healing and tumourigenesis [1].

• Interacts with urokinase plasminogen activating receptor (uPAR), playing a critical role in cancer progression [2, 3].

• No structural information available for αvβ6 integrin to assess its direct interaction with uPAR.

• Hence, we have performed structural analysis of αvβ6•uPAR interactions using model data with docking simulations.

• We have tried to pinpoint αvβ6•uPAR interface, in accord with earlier reports of the β-propeller region of integrin α-chain interacting with uPAR.

Introduction

• Build a comprehensive 3D structural model of integrin αvβ6 heterodimer

• Investigate its distinctive yet descriptive structural properties

• Identify interaction sites at the αvβ6•uPAR interface

• Gain insights into the structural basis of integrin αVβ6•uPAR interactions for its implications as a potential therapeutic target in cancer management

A site for direct integrin αvβ6•uPAR interaction from structural modelling and docking

Gopichandran Sowmya1, 2, Javed Mohammed Khan1, 2 Samyuktha Anand1, Seong Beom Ahn1, Mark S. Baker1, Shoba Ranganathan1, 2, 3

1Department of Chemistry and Biomolecular Sciences and 2ARC Centre of Excellence in Bioinformatics, Macquarie University, Sydney, NSW, Australia

3Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, SingaporeEmail: sowmya.gopichandran@mq.edu.au, shoba.ranganathan@mq.edu.au

Methodology

Interaction sites of αvβ6•uPAR docked complex using PDBsum and comparison with experimental results

Objectives

Preliminary evaluation ► Alignment of the target sequence with template structure

using ClustalX

Figure 1. a. Integrin αvβ6 model depicting the interface area formed by the interacting residues of αv and β6 subunits. b. A graphical representation of αv and β6 interface residues as a function of residue number using ΔASA.

Figure 2. a. Cluster dendogram illustrating the similarity of integrin αvβ6 model to other RGD-binding αβ integrins. b. Heat map depicting the electrostatic similarity of integrin αvβ6 model to other uPAR-binding αβ integrins.

aa residue type Surface(% aa composition)

αV–β6 Interface(% aa composition)

Polar

Neutral 29.64 27.20

Charged +ve 10.40 15.40-ve 13.00 11.80

Total 53.04 54.40Non-polar 46.96 45.60

Table 1: Amino acid (aa) residue composition for the solvent accessible surface and the subunit interface of integrin αvβ6 heterodimeric complex suggest that polar interactions are the predominant forces that drive both αvβ6 subunit interface formation and other protein-protein interactions at the αvβ6 surface.

Figure 3. Integrin αvβ6•uPAR complex with bound uPA and VN. The αvβ6•uPAR docked complex shows that the β-propeller region of α-chain of integrin αvβ6 interacts with the domain III region of uPAR.

Figure 4. The interaction sites of integrin αvβ6•uPAR docked complex. (a) αvβ6 and uPAR rotated by 90° left and right respectively, as indicated. (b) Schematic diagram of αv and uPAR interface. (c) A graphical representation of the uPAR binding residues on integrin αvβ6-uPAR interface obtained from docking simulation as a function of residue number using ΔASA. The residue numbers of the uPAR region identified to bind to αvβ6 integrin by PLA and peptide array experiments are plotted on the x-axis while ΔASA is on the y-axis. The uPAR interface residues and their numbers obtained from the ΔASA analysis of αvβ6-uPAR docked complex are shown. Six out of the 27 residues (identified by PLA and peptide array experiment) are consistent with docking result.

aa residue type Surface(% aa composition)

Polar

Neutral 34.50

Charged+ve 10.70-ve 13.10

Total 58.30Non-polar 41.70

Table 2: Amino acid residue composition for the solvent accessible surface of uPAR shows that 91% of uPAR residues are surface exposed and that the accessible surface is highly polar with predominantly polar interactions.

Conclusions

AcknowledgmentG.S. is grateful to Macquarie University for the award of an iMQRES research scholarship.

• PLA and Peptide array experiments - domain II region (peptides: L172-F189 and C193-E207) & domain III region (peptides: S299-N255, 27aa) of uPAR interact with αvβ6 integrin (unpublished results).

• Six (S229, E230, T248, G249, T250, and E255) out of 27aa in the identified uPAR domain III binding region is consistent with docking data.

• These observations have implications for the abrogation of αvβ6-uPAR interaction as a potential therapeutic target in cancer management, using specific chemical inhibitors as shown for α5β1 [3].

References[1] Bandyopadhyay & Raghavan, Curr.Drug.Targets 2009 10:645-52.[2] Saldanha RG et al., J.Proteome.Res. 2007, 6:1016-1028.[3] Yang J et al., Proc.Natl.Acad.Sci.USA. 2009, 106:17729-34.[4] Chaurasia et al., PLoS One. 2009, 4:e4617.

Results

Data collection►Target sequences: Integrin αv & β6(UniProt: P06756, P18564 )►Templates: integrin αvβ3 (PDB: 3IJE) & αIIbβ3 (PDB: 2KNC)

3D structural modeling, refinement and quality verification

► SAVES and PSVS tools were used for structural verification

Structural analysis ► Interface and surface analysis of integrin αvβ6 calculated as:

► Molecular surface electrostatic potential (MSEP) calculation and comparison with other known integrin crystal structures

Docking of integrin αvβ6 model with uPAR X-ray crystal structure

► Flexible protein-protein docking was carried out with ICM Pro ► β-propeller of αvβ6 - “receptor” & uPAR - “ligand”