BMC Genomics 2009, 10(Suppl 3):S24 http://www.biomedcentral.com/1471-2164/10/S3/S24 form Siaa(2,3)Gal glycosides. The other receptor type contains an a(2,6)-linkage to form Siaa(2,6)Gal. In avian viruses, the preferred HA receptors are of the Siaa (2,3)Gal type, while most human viruses interact with Siaa(2,6)Gal glycoside receptors. Normally, the avian influenza virus H5N1 infects birds rather than humans or other mammalian hosts because their HA binds better to the avian-type Siaa(2,3)Gal glycoside receptor [7,8]. Despite the number of HA variants that have been reported in the protein database [9,10], we do not know enough about the binding mechanism to predict which HA variants can bind efficiently to the human receptor. The most reliable source of information to study the binding mechanism is from X-ray crystal structure. However, only a small number of H5 HA-receptor co- crystals are available in public databases [5]. Another approach to study which HA variants can bind prefer- entially to human receptor is by binding assay experi- ments which systematically screen interactions between HA variants and receptor analogs [11,12]. Nevertheless, to produce and screen many proteins in order to search for human-receptor binding HAs is impractical. Alter- natively, insight into the binding mechanism can be observed by HA protein-receptor simulation to predict the binding potential between different HA variants and human receptor. By this means, one can effectively screen and assign priority to a small number of HA variants for further in vitro experiments. Surveillance of H5 HA mutations over a number of years has led to the discovery of viruses with changes in receptor binding selectivity caused by mutation of the receptor binding site. The H5 HA X-ray structure from A/Duck/Singapore/3/97 (abbreviated as Sing-97) shows preferential binding to Siaa(2,3)Gal receptor, owing to Q222L and G224S mutations [5]. A Sing-97 descendent that infected a human in 2004, A/Thailand/1(KAN-1)/ 2004 (abbreviated as Kan-1), has a single mutation in the HA binding pocket (S129L). This variant, however, still binds preferentially with the avian receptor [13]. A Kan-1 derived strain with further HA mutations L129V and A134V is considered a quasi-species which exhibited higher selectivity toward human cell type receptor [14]. It should be noted that the mutated amino acids considered in the study are not the main binding residues and the mutations do not greatly alter the resulting complex structure. However, some of these mutations do indeed change the receptor type selectivity by contributing to changes in binding mechanism. This modification in host type preference could be caused by differential binding of mutually exclusive conformations of the cell receptor in different HA binding environ- ments. It is hypothesized, that by comparing the conformations of different receptor types bound to different HA binding sites with known binding selectiv- ity, we could distinguish different bound conformations. A recent study by Xu and coworkers [15] has shown that receptor binding preference of different influenza viral types can be modelled by simulation measuring receptor torsion angles. In this study, it is shown how the information from the available structures of HA com- plexed with avian and human receptors can be used to predict the HA binding selectivity from different HA variants in receptor-based conformational analysis by measuring a single torsion angle during Molecular Dynamic simulation. Furthermore, the predictions of receptor preference agree with the available in vitro binding data. Materials and methods Crystallographic datasets and HA variants used for simulation X-ray crystallographic datasets of HA variants complexed with tri-saccharide receptor analogs were obtained from the PDB (Table 1). In the 1JSO dataset, the electron density is ill-defined for the Galb(1,4)GlcNAc sugar residues. Therefore, to allow accurate comparisons between the different crystallographic templates in MD simulations, the structures of the equivalent sugar residues from the receptor in the 1RVZ structural template [16], the closest receptor structure available, were inserted into the 1JSO template. All glycosides were terminated with a methoxy group and were used as the input for molecular dynamics simulations. To prepare HA variants for MD simulation for which no crystal- lographic data are available, homology modelling was performed by three-dimensional alignment with the Table 1: Torsion angles of selected glycosides from protein data bank (PDB) PDB code HA Type Ligand Type Sugar Units 1JSI 1JSO 1RVT 1RVZ 1JSN 1RVX A/Swine/Hong Kong/9/98:H9 A/Duck/Singapore/3/97:H5 A/Swine/Iowa/15/30:H1 A/Puerto Rico/8/34:H1 A/Duck/Singapore/3/97:H5 A/Puerto Rico/8/34:H1 Siaa(2,6)Gal Siaa(2,6)Gal Siaa(2,6)Gal Siaa(2,6)Gal Siaa(2,3)Gal Siaa(2,3)Gal Sia-Gal-GlcNAc-Gal-Glc Sia Sia-Gal-GlcNAc-Gal-Glc Sia-Gal-GlcNAc Sia-Gal-GlcNAc Sia-Gal-GlcNAc Torsion (F) 56.2 - 69.7 67.6 -55.1 -59.1 Resolution (Å) 2.4 2.4 2.4 2.25 2.4 2.2 Citation [5] [5] [16] [16] [5] [16] Page 3 of 9 (page number not for citation purposes)