NMR-STUDIES OF A VIRAL PROTEIN THAT MIMICS THE REGULATORS OF COMPLEMENT ACTIVATION

Vaccinia virus complement control protein (VCP) is a 243-residue prote in that is similar in sequence to the regulators of complement activat ion; its role is to defend the virus against attack by the host comple ment system. A fragment of this protein spanning the two complement pr otein (CP)modules (residues 126 to 243) which make up the C-terminal h alf of VCP has been expressed in Pichia pastoris. A N-15-labelled samp le was purified for the purposes of structure determination and measur ements of dynamics in solution using NMR. Structures were calculated o n the basis of 1767 NMR-derived distance and angle restraints, with a longer than normal high-temperature simulated annealing (SA) protocol which improved convergence. The viral CP-modules are structurally very similar to the 15th and 16th CP-modules of human factor H (fH; averag e r.m.s.d., for invariant Trp and Cys, four pair-wise comparisons, = 1 .2 Angstrom) but less similar to the fifth CP-module of DT (average r. m.s.d. = 2.2 Angstrom). in the VCP fragment, the orientation of one mo dule with respect to the other is clearly defined by the experimental data, and T-1 measurements are consistent with only limited flexibilit y at the module-module interface. The r.m.s.d. over all of the 118 res idues (backbone atoms) is 0.73 Angstrom. The intermodular orientation is better defined than, and significantly different from, that observe d in a CP-module pair from fH (re-calculated using the extended SA pro tocol). Ln VCP the long axis of the second module is tilted by 59(+/-4 )degrees with respect to the first module (50(+/-13)degrees in the fH pair), and twisted with respect to the first module by 22(+/-6)degrees (223(+/-17)degrees in DI). The differences between the human and vira l proteins may be rationalised in terms of the lack of hydrogen-bond s tabilised secondary structure in the N-terminal portion of DI module 1 6, and the number and type of amino acid side-chains which make up the interface. A similar intermodular interface may be predicted between the third and fourth module of human C4 binding protein and, probably, between the third and fourth modules of the guinea pig acrosomal matr ix protein 67; but the formulation of general rules for predicting the structure of interfaces between CP-modules awaits further experimenta l data. (C) 1997 Academic Press Limited.