BMC Genomics 2009, 10(Suppl 3):S24 http://www.biomedcentral.com/1471-2164/10/S3/S24 approximately -55 degrees in the H5 binding pocket, i.e. the receptor is in trans conformation. Meanwhile, Siaa (2,6)Gal exhibits a F angle of approximately +55 degrees, i.e. the receptor is in cis conformation [19]. In other words, Siaa(2,3)Gal seems to have an optimal binding geometry when the Gal and Sia units are bound in the trans conformation, while for the Siaa(2,6)Gal they are instead bound in the cis conformation. Accord- ing to this, we hypothesized that in solution, both conformations are in equilibrium, as suggested by the available crystallographic data [5,16]. Upon binding of HA to the receptor, it is hypothesized that one conformation is favoured; thus, binding drives the equilibrium to this conformation without any molecular readjustment or thermodynamic cost. In order to use the relationships between torsion angles and binding preferences to explain host selectivity of the unknown influenza virus structures, homology model- ling and molecular dynamics (MD) simulations were employed. During each MD simulation, F was mon- itored and interpreted in terms of the binding preference. MD simulations were performed with different combi- nations of avian and human-type receptor analogs with HA variants. The duration of time in which the receptor analog spent in the cis or trans conformation when bound to each HA type varied according to the type of glycosidic linkage and the number of sugar units. Figure 3 presents plots showing different cis/trans conformations. For the binding studies of avian receptor analogs with a(2,3)-linkage, the glycosidic linkage in trans conformation was observed during most of the simulation in all HA systems tested (F angle -55 degrees), albeit with some transient fluctuations to the cis conformation for the Q222L, G224S, and Q222L/ G224S Sing-97 HA variants. In the simulation of human receptor analogs with a (2,6)-linkage, the trans configuration (F = -55 degrees) was observed in the majority during the simulation with some transient fluctuations to the cis conformation when bound to Sing-97, Sing-97 mutants Q222L, G224S and Kan-1 systems,. In contrast, the cis conformation was observed (F = +60 degrees) for the majority of the simulation time when the analogs bound with Puerto-34 human influenza, L129V/A134V Kan-1, and Q222L/ G224S Sing-97 HAs. Discussion From the experimental results, it can be concluded that each of the H5 HA variants exhibited different binding behaviours to different receptor analogs. The available X-ray structures contain only the tri-saccharide part of receptor analogs bound to the binding site, and the last two sugar units are missing or unsolved [5]. Nonetheless, the full-length receptor structures can be constructed using molecular modelling software [26-28]. In this study, modelling was restricted to the tri-saccharide receptor system to minimize the error in the simulations. In our previous work [19], Kan-1 was predicted to bind fairly well to human receptor for a significantly long period; however, this prediction is at variance with in vitro experimental data (Table 2). From the above experimen- tal results, the tri-saccharide simulation model can better estimate the host type binding preference of different H5 HA variants than di-saccharide. The crystallographic data in Ha et al [5] showed that the typical H5 HA binds preferentially to avian receptor with an a(2,3)-linkage in the trans conformation, whereas the typical H1 HA binds preferentially to human receptor with an a(2,6)-linkage in the cis conformation. The simulations presented here predicted that: (i) Sing-97 and Kan-1 bound better to a(2,3) than to a (2,6), since the observed predominant conformation of receptor was trans for both receptor types. (ii) L129V/A134V Kan-1 could bind to both receptor types, since the trans conformation was predominant for a(2,3) binding and the cis conformation for a(2,6) binding. (iii) Q222L, G224S, Q222L/G224S Sing-97 mutants appear to have a weaker preference for a(2,3) than non- mutated Sing-97 because fluctuations from the trans to cis conformation were observed. The a(2,6) simulation of mutated Sing-97 implied that the Q222L/G224L variant had markedly greater binding affinity toward human cell receptor as it bound in cis with a(2,6) all the time. (iv) Human virus Puerto-34 HA bound preferentially with a(2,6), since the observed conformation of receptor was in cis configuration. Prediction of the relative binding selectivity Based on the HA binding conformation preferences from MD simulation (Table 2), predictions of relative binding selectivity (to host-type receptor) can be made as follows. The selectivity toward Siaa(2,3)Gal binding was similar among the three HA variants Puerto-34, Sing97 and Kan-1. The order of selectivity toward Siaa (2,6)Gal binding was Puerto-34 > L129V/A134V Kan-1 ≅ Q222L/G224S Sing-97 > Sing-97 HA ≅ Kan-1. These tendencies were in good agreement with the in vitro binding assays [18,19], in terms of order of preference. Page 5 of 9 (page number not for citation purposes)