Immunophysical properties and prediction of activities for vaccinia virus complement control protein and smallpox inhibitor of complement enzymes using molecular dynamics and electrostatics

Abstract

We present immunophysical modeling for VCP, SPICE, and three mutants using MD simulations and Poisson-Boltzmann-type electrostatic calculations. VCP and SPICE are homologous viral proteins that control the complement system by imitating, structurally and functionally, natural regulators of complement activation. VCP and SPICE consist of four CCP modules connected with short flexible loops. MD simulations demonstrate that the rather complex modules of VCP/SPICE and their mutants exhibit a high degree of intermodular spatial mobility, which is affected by surface mutations. Electrostatic calculations using snapshots from the MD trajectories demonstrate variable spatial distribution of the electrostatic potentials, which suggests dynamic binding properties. We use covariance analysis to identify correlated modular oscillations. We also use electrostatic similarity indices to cluster proteins with common electrostatic properties. Our results are compared with experimental data to form correlations between the overall positive electrostatic potential of VCP/SPICE with binding and activity. We show how these correlations can be used to predict binding and activity properties. This work is expected to be useful for understanding the function of native CCP-containing regulators of complement activation and receptors and for the design of antiviral therapeutics and complement inhibitors.

FIGURE 1

Sequence alignment of VCP, SPICE, VCP2m, VCP3m, and VCP4m. Only mutated amino acids are shown for SPICE, VCP2m, VCP3m, and VCP4m, the remaining amino acids being the same as in VCP. The figure also displays the alignment of the four CCP modules with respect to each other. In each CCP module, there are four conserved cysteines forming two disulfide bridges (marked in figure) and a single conserved tryptophan. The end amino acids of each CCP module are the first and fourth of the conserved cysteines.

FIGURE 2

Ribbon models for the initial minimized crystal structure of VCP (A) and the final (10 ns) structures from the MD simulations of VCP (B), SPICE (C), VCP2m (D), VCP3m (E), and VCP4m (F).

FIGURE 3

Backbone heavy atom RMSDs per CCP module. (A) VCP, (B) SPICE, (C) VCP2m, (D) VCP3m, and (E) VCP4m. The backbone heavy atoms N, C_α, and C were used to calculate the RMSDs from the minimized crystal structures. The stable structures of the last two nanoseconds were used to calculate intermodular skew, tilt, and twist angles (Table 1) and electrostatic potentials (Fig. 6). The MD structures at 10 ns were used to calculate the predicted titration curves and apparent pK_a values (Figs. 8 and 9).

FIGURE 4

Calculated difference in the SASA (ΔSASA) during the MD trajectory. ΔSASA is the SASA of the sum of individual CCPs minus the SASA of the whole protein.

FIGURE 5

Calculated cross correlations of the displacements of pairs of CCP modules for VCP (black), SPICE (white), VCP2m (horizontal fill), VCP3m (downward diagonal fill), and VCP4m (upward diagonal fill).

FIGURE 6

Isopotential surfaces at ±1 k_BT/e, comparing the fluctuations of the spatial distribution of electrostatic potentials for the initial minimized crystal structures and the 8, 9, and 10 ns MD structures. (A and B) VCP, (C and D) SPICE, (E and F) VCP2m, (G and H) VCP3m, and (I and J) VCP4m. The electrostatic potentials were calculated using 0 mM salt concentration (left columns) and 150 mM salt concentration (right columns).

FIGURE 7

Three-dimensional plots of ESI distances for VCP, SPICE, VCP2m, VCP3m, and VCP4m. ESIs were calculated at ionic strengths corresponding to 0 mM (A) and 150 mM (B) concentrations. The plot shows the distinct clustering of the proteins according to the similarity of their electrostatic potentials.

FIGURE 8

Selected titration curves for H-140 of VCP, SPICE, VCP2m, VCP3m, and VCP4m, E-108, E-120 of VCP, and K-108, K-120 of SPICE. The MD structures at 10 ns were used to calculate the predicted titration curves.

FIGURE 9

pH dependence of mean net charge for VCP, SPICE, VCP2m, VCP3m, and VCP4m. (A) pH range 0–16. (B) The vicinity of physiological pH in the range of 6–8, with the mean net charge at pH 7 marked. The MD structures at 10 ns were used to calculate the charges.