BMC Genomics 2009, 10(Suppl 3):S24 http://www.biomedcentral.com/1471-2164/10/S3/S24 most similar structural template (1JSN and 1JSO for Sing-97 and Kan-1 variants; 1RVX and 1RVZ for A/Puerto Rico/8/34 (abbreviated as Puerto-34) variants) using the SWISS-MODEL server [17]. A number of HA strains of known host selectivity were used to test whether Siaa(2,3)Gal would bind to HA with avian receptor preference in trans, and whether Siaa (2,6)Gal would bind to HA with human receptor preference in cis. The MD simulations on seven known HA variants with avian and human receptor types were performed: Sing-97, Puerto-34, Kan-1, mutated Kan-1, and three mutated Sing-97 systems. Among these systems, according to the available binding assay data, two of them, Sing-97 and Kan-1, bind Siaa(2,3)Gal stronger than Siaa(2,6)Gal [18,19]. One of the mutated Sing-97 strains (Q222L/G224S) is known to bind Siaa (2,6)Gal stronger, suggesting that these mutations could be important for changing the receptor preference from avian to human. The other strains except Puerto-34 can equally bind to both types of the receptor. For Puerto-34 there are no experimental data from binding assay, but we assumed strong human receptor binding for this is the strain since it caused a human pandemic [16]. Our prediction scheme was tested on two sets of data. The first set comprised four Kan-1 HA variants each with single mutations M226T, K189G, K218S, and L190P obtained as quasi-species from the source where Kan-1 was found [14]. The reason for testing these variants was to determine whether these strains are potentially harmful, i.e. stronger binding to human receptor than Kan-1. The second set contained three mutated Puerto- 34 HA variants, Q222L, G224S, and Q222L/G224S (H5 numbering). These variants were chosen to determine what effect these mutations had on a different HA type (H1) to Sing-97 (H5). Molecular dynamic simulations To permit comparison with previous experimental results [19], all the simulations were done using the same Glycam04 [20] parameters for receptor and AMBER 2003 force field for protein. Initial structure was first solvated using the TIP5P water model [21] in the truncated octahedron box. Energy minimization was then performed to relieve bad contacts caused by unreasonable distances in the structure by keeping the protein and receptor restrained. The whole system was relaxed at 0 K with 10 Å non-bonded cutoff. The temperature of the system was then set to 300 K and equilibrated for 100 picoseconds with weak restraint on both receptor and protein, where bonds involving hydrogen are constrained using the SHAKE algorithm [22]. Torsion angles (F) were monitored throughout the simulation to determine the conformation of the glyco- sides. The F angle is defined as the angle between the O6-C2 bond of Sia and the glycosidic bond of Gal units (Figure 1). Note that the F angle defined by Xu and colleagues [15] refers to a different plane of rotation of the receptor. The F torsion angle described in this study was not considered in Xu et al. To determine the binding preference, the F angle was monitored in order to reflect the receptor type selectivity. The 3 nanosecond production run was performed at a constant temperature and pressure with 0.002 picose- conds time step (without restraining) using the SANDER module in the AMBER9 program [23]. The structures stabilized after 1.5 nanoseconds as shown in Figure 2. The highest degree of fluctuation was observed for residues located at the terminal chains of the structures. These residues were inserted in HA chain B and were left unrestrained during the simulations; however, they did not disturb the binding site (average root mean square deviation (RMSD) between residues 30 and 310 was less than 0.5 Å). The utility programs, Xmgrace [24], and VMD [25] were used to visualize and render all the figures presented in this paper. Results Data from available co-crystal structures of cell receptor analog and HA were used to establish relationships between bound receptor conformation and host type preferences. Comparison between these structures revealed that the F torsion angle, has different values for Siaa(2,3)Gal and Siaa(2,6)Gal binding (Table 1). The observed values show Siaa(2,3)Gal has a F angle Figure 2 Six nanosecond RMSD plots of three simulated HA systems: Sing-97 (black), L129V/A143V Kan-1(red), and Puerto-34 (green). Page 4 of 9 (page number not for citation purposes)