THE MECHANISM OF BINDING STAPHYLOCOCCAL PROTEIN-A TO IMMUNOGLOBIN-G DOES NOT INVOLVE HELIX UNWINDING

Structural changes in staphylococcal protein A (SpA) upon its binding to the constant region (Fc) of immunoglobulin G (IgG) have been studie d by nuclear magnetic resonance and circular dichroism (CD) spectrosco py. The NMR solution structure of the engineered IgG-binding domain of SpA, the Z domain (an analogue of the B domain of SpA), has been dete rmined by simulated annealing with molecular dynamics, using 599 dista nce and dihedral angle constraints. Domain Z contains three alpha-heli ces in the polypeptide segments Lys(7) to His(18) (helix 1), Glu(25) t o Asp(36) (helix 2), and Ser(41) to Ala(54) (helix 3). The overall cha in fold is an antiparallel three-helical bundle. This is in contrast t o the previously determined X-ray structure of the similar SpA domain B in complex with Fc, where helix 3 is not observed in the electron de nsity map [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370], but sim ilar to the solution NMR structure of domain B, which is also a three- helical bundle structure [Gouda, H., et al. (1992) Biochemistry 31, 96 65-9672]. In order to characterize possible secondary structural chang es associated with IgG binding, far-UV CD spectra were collected for t he Z domain, an engineered repeat of this molecule (ZZ), recombinant F c from IgG subclass 1 (Fc(1)), recombinant Fc from IgG subclass 3 (Fc( 3)), and mixtures of Z/Fc(1), Z/Fc(3), ZZ/Fc(1), and ZZ/Fc(3) Fc(3) wa s included as a control for possible changes of the CD spectrum in the mixture of noncomplexed molecules, since SpA is known not to bind Fc( 3). From these CD spectra, it was concluded that the third alpha-helix in Z is not disrupted in its complexes with Fc(1). Similar results we re obtained for the ZZ molecule. However, in both Z and ZZ there are s ome perturbations in CD spectra at high energy wavelengths (i.e., lamb da < 215 nm) accompanying complex formation. On the basis of the combi ned CD and NMR results, as well as previously described binding studie s of Z mutant proteins to Fc(1), we conclude that the Z domain maintai ns its three-helical bundle structure in the Z-Fc complex, though ther e may be a small structural change involved in the binding mechanism.