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.

Enhancing the population of the encounter complex affects protein complex formation efficiency

“…All rights reserved measured by observing ET from Cc(Fe 2+ ) to CcP compound I (CpdI), which is formed after reaction with hydrogen peroxide, example data are given in Figure S3. The ET rate constant is high, so the observed second order rate constant is a lower-limit estimate of the association rate constant, as explained in detail in a previous paper [24], see also equation 1 in the Materials and Methods. The association rate constant for wt Cc to CcP_A and CcP_B is strongly dependent on the ionic strength (Figure 5, data reproduced from ref [23,24]), due to the favorable electrostatic interactions between Cc and CcP [29][30][31][32][33].…”

“…The ET rate constant is high, so the observed second order rate constant is a lower-limit estimate of the association rate constant, as explained in detail in a previous paper [24], see also equation 1 in the Materials and Methods. The association rate constant for wt Cc to CcP_A and CcP_B is strongly dependent on the ionic strength (Figure 5, data reproduced from ref [23,24]), due to the favorable electrostatic interactions between Cc and CcP [29][30][31][32][33]. Following the same approach, the ka was measured for Cc R13A and CcP_A or CcP_B.…”

“…The kinetic curve measured at the stopped flow is recreated summing the contributions of each species to the absorbance at 416 nm according to the Beer-Lambert law: [24]. The values of k(II), k(III) and k(IV) were independently changed manually until the simulation was considered a good fit as judged by visual observation.…”

“…Arg13 has been shown to be a hot-spot in the interactions of the stereospecific complex and its mutation to Ala shifts the balance toward the encounter state and reduces the affinity by 30 fold [25,26]. This mutant was used to study the interaction with native CcP (CcP_A) and a variant of CcP with four Lys residues mutated to Glu on the side of CcP relative to the stereospecific binding site, resulting in a charge change of -8 (CcP_B) (CcP_B, Figure 1) [23,24]. Chemical shift perturbation (CSP) analysis and paramagnetic relaxation enhancement (PRE) experiments show that the complex is highly dynamic and that Cc visits the new negative patch in CcP_B.…”

In vitro evolution of uracil glycosylase towards DnaKJ and GroEL binding evolves different misfolded states

“…GroEL and DnaKJ chaperones recognize exposed motives at the surface of misfolded proteins that are mostly hydrophobic, which in the case of DnaK are also flanked by positive charges. Noticeably, these can be naturally enriched in mutants through a natural bias in evolution: the codon usage bias dictates that random mutagenesis should more frequently generate spontaneously sticky mutations, mainly hydrophobic and also positive charges that can interact with the negative charges that are often on the surface of native soluble proteins (43) (Figure 6A and B and Table S4). A protein can explore a larger mutational space and even include promiscuity-enhancing mutations due to the chaperones acting as enzymes that can iteratively unfold misfolded but not native protein conformations (30).…”