N9 Neuraminidase Complexes with Antibodies NC41 and NC10: Empirical Free Energy Calculations Capture Specificity Trends Observed with Mutant Binding Data

Abstract

X-ray crystallographic coordinates of influenza virus N9 neuraminidase complexed with monoclonal antibodies NC41 and NC10 [Tulip et al. (1992) J. Mol. Biol. 227, 122-148] served as a starting point for calculations aimed at estimating free energy changes (delta G) of complex formation between the two antibodies and the neuraminidase. Using an empirical function incorporating hydrophobic, electrostatic, and conformational entropy effects, we estimated contributions individual neuraminidase residues make to complex formation (delta G(residue)) and compared the calculated values to experimentally measured differences in antibody binding between the wild-type and mutated neuraminidases [Nuss et al. (1993) Proteins 15, 121-132; calculations done without prior knowledge of the experimental data]. A good correspondence was found between the calculated delta G(residue) values and the mutant binding data in that side chains with large calculated delta G contributions (delta G(residue) < -1 kcal/mol) lie at sites of mutation which cause a marked reduction in antibody binding, and side chains for which delta G(residue) > -1 kcal/mol are sites at which a mutation does not have a marked effect on binding. Because most of the delta G(residue) < -1 kcal/mol side chains also make hydrogen bonds/salt bridges with the antibody, the correspondence of the effect of antibody binding with these electrostatic interactions (18 out of 27 for NC41 and, tentatively, 5 out of 7 for NC10) is about as good as that with predicted energetic residues. All the delta G(residue) < -1 kcal/mol neuraminidase side chains cluster around the most protruding surface regions and are thus spread over different epitope segments. Surprisingly, different residues were found to make the most critical contributions to the NC41 and NC10 complex stabilities despite the fact that the NC41 and NC10 antigenic epitopes overlap, having approximately 70% of surface residues in common. It is thus possible, for two different antibodies, to recognize the same protein surface in strikingly different ways. As only a fraction of the neuraminidase residues appear to make large contributions to antibody binding, the results also support the hypothesis of a "functional" epitope in antigen-antibody interactions. Positive trends between both backbone rigidity and residue accessibility in the complexed state, and contributions of these residues to binding, were also observed for the NC41 complex.

Electrostatic complementarity at protein/protein interfaces 1 1Edited by B. Honig

“…Of these, tern N9 Lys432 and NC41 V H Glu96 and Asp97 are in the region that overlaps with the NC10 epitope, and correspond to the segments of the surface that dominate the electrostatic complementarity of the overlapping epitope ( Figure 3). In the whale N9±NC10 complex, Tulip et al (1994) concluded that whale N9 Lys432 and NC10 V H Asp56 contributed most to binding. Both of these are in the region that overlaps with the NC41 epitope, and again these correspond to the segments of the surface that dominate the electrostatic complementarity of the overlapping epitope.…”

“…In particular, Tulip et al (1994) calculated the (electrostatic) contribution to the total ÁG of binding made by each NA residue in the interface and concluded that different residues made the greatest contributions to the NA ± NC41 and NA ±NC10 complex stabilities. They concluded that in the tern N9 ±NC41 complex, tern N9 Lys432 and Lys463 and NC41 V H Glu56, Glu96 and Asp97 contributed most to binding.…”

Analysis of protein-protein interactions and the effects of amino acid mutations on their energetics. The importance of water molecules in the binding epitope

“…These studies ®nd that the strength and speci®city of protein-protein associations is the result of a wide variety of interactions within the binding interface (Novotony et al, 1989;Cunningham & Wells, 1993;Tulip et al, 1994). While the energetics of these interactions are known to arise from hydrogen bonding, hydrophobicity, ion pairing and surface complementarity, their relative contributions to binding are known to be quite variable (Clackson & Wells, 1995).…”

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection

“…While a recombinant HA-based A(H7N9) vaccine has been developed and clinically evaluated (4), the contribution of NA immunity against A(H7N9) infection has been less explored. The antigenic structure of N9 has been described in studies of A/tern/Australia/G70C/75 (G70C, H11N9) and A/whale/1/84 (H13N9), with X-ray crystallography of N9 and monoclonal antibody (MAb) complexes defining epitopes that surround the enzyme active site (17)(18)(19)(20). However, the antigenic characteristics of the NA of recent A(H7N9) viruses are poorly defined.…”