The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed

Abstract

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

Figure 1

CcP variants. (a) Schematic representation of the complexes formed by Cc and the CcP variants. Cc is represented as a blue circle bound at the stereospecific binding site, and the CcP variants are represented as empty circles with the negative charges on the surface indicated as red dashes. (b) Electrostatic potential plotted on the surface of the CcP variants ranging from −5 (red) to 5 kcal/e° (blue) at an ionic strength of 120 mM. (c) Structure of CcP (green ribbon, red heme) surrounded by the centers of the mass of Cc in the ensemble of encounters of the Cc:CcP_A (cyan), Cc:CcP_B (magenta), Cc:CcP_C (yellow), Cc:CcP_D (salmon), Cc:CcP_E (gray), and Cc:CcP_F (purple) complexes as obtained from rigid body Monte Carlo simulations. (d) Energy distribution of the encounter complexes between Cc and the CcP variants as obtained from rigid body Monte Carlo simulations. The inset shows the entire distribution for CcP_F using a different vertical scale.

Figure 2

Comparison between fitting and simulation of the reaction performed by Cc and CcP_A at an ionic strength of 292 mM measured at the stopped flow. A gray area covers the first part of the measured data affected by a stopped flow artifact and thus excluded from the analysis. (a) Fitting of the fast decay to eq 1 (see Materials and Methods). The data are shown as a solid black line, and the fit is represented by the red line. The extrapolation of the fit is shown as a dashed line. (b) Simulation (red line) of reactions II–IV to fit the same data (black) as in panel a (left). Concentrations over time of all of the species involved in the Cc:CcP cycle as derived from the simulation (right).

Figure 3

Rates of association (k_a) between Cc and the CcP variants. The k_a values, plotted as a function of the square root of the ionic strength, were obtained from the simulations of the stopped flow kinetics. Errors were calculated as the standard deviation between replicates and simulations performed at different CcP concentrations (see Materials and Methods for details).