Model of Molecular Interaction Between of Scfv and HA

Supplemental data 3

Model of molecular interaction between of scFv and HA

The software Discovery Studio 3.5 (Accelrys Inc, California, USA) was employed to create HA-antibody complex model. The antibody 3JA18 was created by using PDB3JUY[1] as the template structure with sequence replaced with that of 3JA18. To attain the refined structure, Smart Minimizer protocol, 1000 steps of Steepest Descent with a RMS gradient tolerance of 3 followed by Conjugate Gradient minimization with a RMS gradient tolerance of 0.1, was performed with fix-backbone constraints.

The crystal structure of a H5N1 influenza virus hemagglutinin, PDB2FK0, composed of trimers of HA1 and HA2 and the post-fusion structure of HA2 trimers (a.a. 23-185), PDB1QU1, were adopted for the antibody-protein docking. Dock Proteins (ZDOCK) protocol [2] was carried out by using the generated scFv 3JA18 as the ligand protein. 2000 poses of the HA-scFv 3JA18 complex were obtained. RDOCK (Refine Docked Proteins) protocol refinedZDOCKpredictions by energy minimizationand then scored the refined poses by energy analysis [3-5]. The HA-ligand interactions were analyzed by DS Analyze Protein Interface protocol with surface area probe radius of 0.6 Å, maximum hydrogen bond distance of 2.5 Å and maximum salt bridge distance of 5 Å.The forcefield of CHARMm and dielectric constant of 4 were employed for the calculations.

The analyzed results of scFv 3JA18-HA trimer complex showed that the epitope targeted by 3JA18 may consist in the base of HA trimer (Supplemental data 2A and 2B). The docking results also exhibited that scFv 3JA18 has affinity to HA1 near the HA cleavage site (321-RNSP-324) that may interfere with cleavage by the cellular protease and render HA inactivated [6]. According to PDB2FK0 model, the binding residues within scFv 3JA18 contain the VH-CDR2 segment (61-TISSLGAITR-70), the main antigen-targeting site, which binds to the fusion peptide (from I6 to G16) and the heptad repeat (HR) region of HA2, thereby impeding the membrane fusion between viral and host membranes [6,7]. Since FP and HR regions are highly conserved, the results suggest that scFv 3JA18 antibody is cross-strain and cross-subtypic.

On the other hand, as the post-fusion structure (PDB1QU1, a HA2 trimer) was used for the receptor protein (Supplemental data 2C and 2D), scFv 3JA18 bound to twostrands of a HA2 trimer, of which the binding sites contained the low-pH induced helixarea (Region B as indicated in Supplemental data 3) [8].In this model, VH-CD2 segment was not shown in the binding interface for clarity.

In the models shown below, HA1 was coded in blue, the 3 strands of HA2 were in red, pink and purple, respectively, and scFv 3JA18 was in green. For both models, (A,B) displayed the entire complex structurewith residues 153-156 highlighted by yellow CPK; (C,D) detailed the intermolecular interactions of participating residues. Hydrogen bondswere indicated by green dashed lines. Pi-interaction(a non-covalent interaction involving -systems) and salt bridge (involvinghydrogen bonding and electrostatic interactions) were marked as orange and yellow lines, respectively.

References

1. Clark KR, Walsh ST (2009) Crystal structure of a 3B3 variant--a broadly neutralizing HIV-1 scFv antibody. Protein Sci 18 (12):2429-2441. doi:10.1002/pro.255

2. Chen R, Weng ZP (2002) Docking unbound proteins using shape complementarity, desolvation, and electrostatics. Proteins 47 (3):281-294. doi:Doi 10.1002/Prot.10092

3. Chen R, Li L, Weng ZP (2003) ZDOCK: An initial-stage protein-docking algorithm. Proteins 52 (1):80-87. doi:Doi 10.1002/Prot.10389

4. Chen R, Mintseris J, Janin J, Weng Z (2003) A protein-protein docking benchmark. Proteins 52 (1):88-91. doi:10.1002/prot.10390

5. Li L, Chen R, Weng Z (2003) RDOCK: refinement of rigid-body protein docking predictions. Proteins 53 (3):693-707. doi:10.1002/prot.10460

6. Sriwilaijaroen N, Suzuki Y (2012) Molecular basis of the structure and function of H1 hemagglutinin of influenza virus. Proc Jpn Acad Ser B Phys Biol Sci 88 (6):226-249. doi:DN/JST.JSTAGE/pjab/88.226 [pii]

7. Chambers P, Pringle CR, Easton AJ (1990) Heptad Repeat Sequences Are Located Adjacent to Hydrophobic Regions in Several Types of Virus Fusion Glycoproteins. Journal of General Virology 71:3075-3080. doi:Doi 10.1099/0022-1317-71-12-3075

8. Bullough PA, Hughson FM, Skehel JJ, Wiley DC (1994) Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 371 (6492):37-43. doi:10.1038/371037a0