Prediction of avian Influenza A binding preference to

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

Introduction Avian influenza H 5 N 1 Chain A of H 5 HA (A/Duck/Singapore/3/97 HA) Host cell receptor TYR 191 SER 129 GLN 222 TYR 91 TRP 149 SER 133 Hemagglutinin protein Ha, Y. , et al. Proc. Natl. Acad. Sci. USA 98: 11181 -11186

Introduction Avian Influenza A virus Avian receptor Φ = -55 degree Tran conformation Human receptor Φ = 55 degree Cis conformation HA Mutation potentially changing the receptor binding preference Hemagglutinin Protein Mutated KAN-1 Mutated Sing-97 Viseshakul N et al. Virology 2004, 328, 169 -

Objectives • 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

Material & Method X-ray crystal structures 1 JSO template for Sing-97 and Kan 1 variant 1 RVZ template for Puerto 34 variant host cell receptor X-ray structure or 3 D- homology models of HA protein HA complex MD simulation Known HA variants * : • Sing-97 • Puerto-34 (human flu virus) • Sing-97 variant (Q 222 L, G 224 S, Q 222 L/G 224 S) • Kan-1 • Double KAN-1 mutant (L 129 V/A 134 V) Prediction for HA mutants • Single Kan-1 HA : M 226 T, K 189 G, K 218 S and L 109 P • Puerto-34 HA variants : Q 222 L, G 224 L, Q 222 L/G 224 S Observed torsion angle of host cell receptor Binding preference

Results & Discussion Comparison of observed torsion angle from binding simulation cis Human receptor • Puerto-34 bind preferentially with human type * * • Sing-97 bound better to Avian > human • Q 222 L/G 224 L sing-97 bind preferentially with human type • Kan-1 bind better to Avian > human • L 129 V/A 134 V Kan-1 bind preferentially with human type Puerto-34 tran Avian receptor Sing-97 cis Q 222 L Sing-97 G 224 S Sing-97 Q 222 L/G 224 S Sing-97 tran Kan-1 cis Kan-1 L 129 V/A 134 V KAN-1 tran *Harvey R et al. J Virol 2004, 78: 502 -507.

Results & Discussion MD predicted receptor type selectivity of HAs compared with data from published experimental assays Order of binding selectivity to human receptor flu viruses Puerto-34 > L 129 V/A 134 V Kan-1≅ Q 222 L/G 224 S Sing-97 > Sing-97 ≅ Kan-1 Tested HA variants Simulation in vitro Puerto-34 +++ Sing-97 ++ + Q 222 L Sing-97 ++ ++ G 224 S Sing-97 + ++ Q 222 L/G 224 S Sing-97 +++ Kan-1 + + L 129 V/A 134 V Kan-1 +++ +++ most preferred, ++ moderately preferred, + least preferred

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

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

Conclusions Binding Preference Human receptor associates with positive (Cis conformation) +Φ angle Avian-type receptor associates with negative -Φ angle (Tran conformation) Using Molecular dynamics simulation to observe torsion angles “in silico Mutagenesis”

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

THANK YOU