Prediction of avian Influenza A binding preference to human receptor using conformational analysis of receptor bound to hemagglutinin Wanwimon Mokmak, M.Sc. Biostatistics and Informatics Laboratory Genome Institute, BIOTEC, Thailand

TYR191 SER129 GLN222 TRP149 TYR91 SER133 Ha, Y., et al. Proc.Natl.Acad.Sci.USA 98: Chain A of H5 HA (A/Duck/Singapore/3/97 HA) Avian influenza H5N1 Hemagglutinin protein Host cell receptor Introduction

Hemagglutinin Protein potentially changing the receptor binding preference HA Mutation Φ = 55 degree Cis conformation Φ = -55 degree Tran conformation Introduction Viseshakul N et al.Virology 2004, 328, Avian Influenza A virus Mutated KAN-1 Mutated Sing-97 Avian receptor Human receptor

To predict the HA binding selectivity from different HA variants in receptor-based conformational analysis by measuring a single torsion angle (Φ) during molecular dynamic simulation Objectives

Material & Method X-ray crystal structures 1JSO template for Sing-97 and Kan1 variant 1RVZ template for Puerto 34 variant host cell receptorX-ray structure or 3D- homology models of HA protein HA complex MD simulation Binding preference Observed torsion angle of host cell receptor Known HA variants * : Sing-97 Puerto-34 (human flu virus) Sing-97 variant (Q222L, G224S, Q222L/G224S) Kan-1 Double KAN-1 mutant (L129V/A134V) Prediction for HA mutants Single Kan-1 HA : M226T, K189G, K218S and L109P Puerto-34 HA variants : Q222L, G224L, Q222L/G224S

Comparison of observed torsion angle from binding simulation Results & Discussion Puerto-34 Sing-97 Kan-1 tran cis tran cis tran cis Sing-97 bound better to Avian > human Q222L/G224L sing-97 bind preferentially with human type Kan-1 bind better to Avian > human L129V/A134V Kan-1 bind preferentially with human type Puerto-34 bind preferentially with human type Sing-97 Q222L Sing-97 G224S Sing-97 Q222L/G224S Sing-97 Kan-1 L129V/A134V KAN-1 * Harvey R et al. J Virol 2004, 78: Auewarakul P. et al. J Virol 2007, 81: * Human receptor Avian receptor *

Results & Discussion MD predicted receptor type selectivity of HAs compared with data from published experimental assays Order of binding selectivity to human receptor for flu viruses Puerto-34 > L129V/A134V Kan-1 ≅ Q222L/G224S Sing-97 > Sing-97 ≅ Kan-1 Tested HA variantsSimulationin vitro Puerto Sing Q222L Sing-97++ G224S Sing Q222L/G224S Sing Kan-1++ L129V/A134V Kan most preferred, ++ moderately preferred, + least preferred

Simulation results of four Kan-1 HA mutant variants binding to Human receptor Prediction of the Relative Binding Selectivity Results & Discussion Tran Cis

Simulation results of four Puerto-34 HA mutants Prediction of the Relative Binding Selectivity Results & Discussion Tran Cis

Human receptor associates with positive +Φ angle (Cis conformation) Avian-type receptor associates with negative -Φ angle (Tran conformation) Using Molecular dynamics simulation to observe torsion angles Conclusions

Dr. Sissades Tongsima Dr. Chak Sangma Ms.Nipa Jongkon Ms.Daungmanee Chuakheaw Mr. Anunchai Assawamakin Dr. Philip J. Shaw Dr. Jittima Piriyapongsa Acknowledgements National Center for Genetic Engineering and Biotechnology (BIOTEC) Kasetsart University

THANK YOU

X-ray structure to prepare HA variant for MD simulation: 1JSN & 1JSO template for Sing-97 and Kan1 variants 1RVX & 1RVZ template for Puerto 34 HA variants * : Sing-97 Mutated Singe-97 (Q222L, G224S, Q222L/G224S) KAN-1 mutated KAN-1 (L129V/A134V) Puerto-34 *available binding assay Prediction model: Kan-1 HA variant : M226T, K189G, K218S and L109P Puerto-34 HA variant : Q222L, G224L, Q222L/G224S HA variants and prediction HA variants used for simulation Material & Method Using the SANDER module in the AMBER9 program

Six nanosecond RMSD plots of three simulation HA system Results & Discussion

Molecular dynamic simulations Glycam04 parameters for receptor AMBER 2003 force field for protein using the TIP5P water model in the truncated octahedron box Using the SANDER module in the AMBER9 program To determine the binding preference Φ angle approximately -55 degreesΦ angle approximately +55 degrees Materials and Methods

Results & Discussion Figure 3. Comparison of observed torsion angle (Φ) from the binding simulation between HA variants and avian and human type receptor analog. Φ Was monitored and interpreted in term of the binding preference Siaα(2,3)GalSiaα(2,6)Gal Puerto-34 Results & Discussion

Siaα(2,3)GalSiaα(2,6)Gal Comparison of observed torsion angle (Φ) from the binding simulation between HA variants and avian and human type receptor analog. Sing-97 Results & Discussion

Figure 3. Comparison of observed torsion angle (Φ) from the binding simulation between HA variants and avian and human type receptor analog. Siaα(2,3)GalSiaα(2,6)Gal Kan-1 Results & Discussion

PDB code HA TypeLigand TypeSugar Units Torsion (Φ) Resolution (Å) 1JSIA/Swine/Hong Kong/9/98:H9 Siaα(2,6)Ga l Sia-Gal- GlcNAc- Gal-Glc JSOA/Duck/Singapore/3/97:H 5 Siaα(2,6)Ga l Sia-2.4 1RVTA/Swine/Iowa/15/30:H1Siaα(2,6)Ga l Sia-Gal- GlcNAc- Gal-Glc RVZA/Puerto Rico/8/34:H1Siaα(2,6)Ga l Sia-Gal- GlcNAc JSNA/Duck/Singapore/3/97:H 5 Siaα(2,3)Ga l Sia-Gal- GlcNAc RVXA/Puerto Rico/8/34:H1Siaα(2,3)Ga l Sia-Gal- GlcNAc Torsion angles of selected glycosides from PDB Results & Discussion