The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of −1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic “hot-spots”. Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic “hot-spots” at the two functional sites of C3d, while the surface of CR2 lacks electrostatic “hot-spots” despite its excessively positive nature. We propose that the electrostatic “hot-spots” of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.

Author Summary

Complement fragment C3d is a thioester-containing protein that is a key component/domain in the complement system, an ancient line of defense, due to its ability to covalently attach to pathogen cell surfaces, such as bacteria. As the immune system evolved in complexity, from acellular defense mechanisms to multicellular systems with memory, so has the function of C3d. In humans, but not lower species such as invertebrates, C3d attached to pathogen surfaces binds B-cell co-receptor CR2, in conjunction with an antibody/antigen complex, forming a link between the innate and adaptive immune systems. The C3d-CR2 interaction ultimately increases B-cell sensitivity to the C3d tagged pathogen by 1,000–10,000 fold, and is known to be driven by electrostatic forces. Since electrostatics are crucial to the C3d-CR2 interaction, it is likely that probing the evolution of the electrostatics of C3d and CR2 will provide insight into this gained function. To this end, we employ a novel computational approach for identifying the electrostatic “hot-spots” of C3d and CR2, which are produced by clusters of like-charged residues found on the surface of the protein. Electrostatic “hot-spots” are often evolutionarily favored and in this study provide new insight into the evolution of C3d in its role in a link between innate and adaptive immunity.

The V3 loop of the glycoprotein 120 (gp120) is a contact point for cell entry of HIV-1 leading to infection. Despite sequence variability and lack of specific structure, the highly flexible V3 loop possesses a well-defined role in recognizing and selecting cell-bound coreceptors CCR5 and CXCR4 through a mechanism of charge complementarity. We have performed two independent molecular dynamics (MD) simulations to gain insights into the dynamic character of two V3 loops with slightly different sequences, but significantly different starting crystallographic structures. We have identified highly populated trajectory-specific salt bridges between oppositely charged stem residues Arg9 and Glu25 or Asp29. The two trajectories share nearly identical correlated motions within the simulations, despite their different overall structures. High occupancy salt bridges play a key role in the major cross-correlated motions in both trajectories, and may be responsible for transient structural stability in preparation for coreceptor binding. In addition, the two V3 loops visit conformations with similarities in spatial distributions of electrostatic potentials, despite their inherent flexibility, which may play a role in coreceptor recognition. It is plausible that cooperativity between overall electrostatic potential, charged residue interactions, and correlated motions could be associated with a coreceptor selection and binding.

Background

The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes.

Results

Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N6X7T8|S8X9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution.

Conclusions

We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3 loop with coreceptors CCR5/CXCR4, whereas the charge distribution contributes to the specific short-range interactions responsible for the formation of the bound complex. We also propose a scheme for coreceptor selectivity based on the sequence glycosylation motif, the 11/24/25 rule, and net charge.

Electrostatic interactions are ubiquitous in proteins and dictate stability and function. In this review, we discuss several methods for the analysis of electrostatics in protein–protein interactions. We discuss alanine-scanning mutagenesis, Poisson–Boltzmann electrostatics, free energy calculations, electrostatic similarity distances, and hierarchical clustering of electrostatic potentials. Our recently developed computational framework, known as Analysis of Electrostatic Similarities Of Proteins (AESOP), incorporates these tools to efficiently elucidate the role of electrostatic potentials in protein interactions. We present the application of AESOP to several proteins and protein complexes, for which charge is purported to facilitate protein association. Specifically, we illustrate how recent work has shaped the formulation of electrostatic calculations, the correlation of electrostatic free energies and electrostatic potential clustering results with experimental binding and activity data, the pH dependence of protein stability and association, the design of mutant proteins with enhanced immunological activity, and how AESOP can expose deficiencies in structural models and experimental data. This integrative approach can be utilized to develop mechanistic models and to guide experimental studies by predicting mutations with desired physicochemical properties and function. Alteration of the electrostatic properties of proteins offers a basis for the design of proteins with optimized binding and activity.

Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction.