BMC Genomics 2009, 10(Suppl 3):S24 http://www.biomedcentral.com/1471-2164/10/S3/S24 Table 2: MD predicted receptor type selectivity of HAs compared with data from published experimental assays Simulation in vitro a2,3 +++* +++* +* +++* +++ ++ ++ ++ +++ +++ a2,6 +++ ++ + +++ ++ ++ + +++ + +++ a2,3 +++ ++ ++ + +++ +++ a2,6 +++ + ++ ++ +++ + +++ Citation [16] - - - [5] [29] [29] [29] [19] [19] Tested HA variants Puerto-34 Q222L Puerto-34 G224S Puerto-34 Q222L/G224S Puerto-34 Sing-97 Q222L Sing-97 G224S Sing-97 Q222L/G224S Sing-97 Kan-1 L129V/A134V Kan-1 - no data available, +++ most preferred, ++ moderately preferred, + least preferred. * - Siaa(2,3)Galb(1,4)GlcNAc not Siaa(2,3)Galb(1,3)GlcNAc. Therefore, the duration of the cis conformation during the simulation may be correlated with the selectivity of the docked HA. According to our prediction scheme, the L129V/A134V Kan-1 variants, i.e. mutations M226T, K189G, K218S, and L190P, may have increased the selectivity slightly toward human receptor, since the receptor was present in cis conformation for some of the simulation (Figure 4). The results from the three mutated Puerto-34 systems also showed some changes in their binding behaviors compared to non-mutated Puerto-34 (Figure 5). The two single mutations, Q222L and G224S cause a loss in human receptor affinity as shown by fluctuations to the Figure 4 Simulation results of four mutated-Kan-1 HA systems. M226T, K189G, K218S and L190P binding to human receptor a(2,6) analog. trans-conformation, while the double mutation Q222L/ G224S maintained its preference for human receptor as the bound a(2,6) glycosides were in the cis conforma- tion. For the a(2,3) receptor, all the Puerto-34 systems except for G224S appear to interact weakly since the receptor is in the cis-conformation. The trans conforma- tion is observed for G224S, although the average torsion angle is increased from -50 to -30 degrees, suggesting that it may not be optimal for binding. The results show that mutations at residues 222 and 224 have minor impact on host preference for Puerto-34 HA Conclusion We have shown that our cis-trans conformational analysis scheme could predict the host type selectivity of HA variants. Our cis-trans conformation hypothesis also worked well under another HA system where it predicted the cis conformation and revealed the similar mechanism in Puerto-34 simulation. The binding patterns and mechanisms of the adopted receptor model, Siaa(2,3)Gal and Siaa(2,6)Gal, to wild-type and mutated Kan-1 HA, and Sing-97 HA were proposed. The results could be used to explain why the L129V/ A134V Kan-1 and Q222L/G224S Sing-97 could bind better to human receptor analog in in vitro assays. The underlying proposed mechanism that made H5 bind to human host without mutation at residue 222 or 224 involved the interaction between residue 134 side-chain and Gln222. It is proposed that mutations change the HA binding preference from Siaa(2,3)Gal to Siaa(2,6) Gal. Our study also suggested that even mutations outside of key binding residues [29], e.g. residue 222 or 224, have consequences on altering receptor type and should not be ignored. Furthermore, our procedure is useful for predicting host type, which can be tested by in vitro binding assays. Page 7 of 9 (page number not for citation purposes)