(781g) The Role of Electrostatics in the Function of a Protein Link Between Innate and Adaptive Immunity

The protein C3d is a proteolytic fragment of C3, the converging protein of three activation pathways of the complement system. Upon cleavage and activation of C3, the fragment C3b covalently attaches to pathogen or damaged host cell surfaces through its thioester domain (TED), acting as (i) an opsonin for recognition of C3b-decorated surfaces by phagocytes, and (ii) as a component of surface-bound convertase complexes that amplify C3 cleavage in a C3b production feedback loop [1]. Further cleavage steps inactivate C3b by converting to C3d, a surface-bound standalone TED. C3d is a biomarker indicating that complement system has been activated on a particular surface. C3d is also a link between innate (complement system based) and adaptive (antigen-antibody based) immunity, through the formation of the C3d-complement receptor 2 (CR2) complex, which is part of the B cell receptor (i.e. antibody) â?? coreceptor (i.e. CR2) complex. This complex cross-links antigens with B cells and augments antibody production by up to four orders of magnitude.

The association between C3d and CR2 is a two-step process, driven by the long-range electrostatic attraction of protein macrodipoles (recognition step) and the formation of a stable bound complex based on favorable short-range polar and nonpolar pairwise amino acid interactions (binding step) (see [2] and references therein). This model is based on computational studies, such as molecular dynamics simulations, electrostatic calculations, theoretical mutagenesis and alanine scans, and free energy analyses. Also, Brownian dynamics simulations demonstrate that electrostatic steering accelerates the formation of an encounter complex during recognition of C3d and CR2, which is ionic strength dependent [3]. In all cases, the computational data have been compared and are in agreement with existing experimental binding, mutagenesis, and ionic strength studies.

The electrostatic interaction functionality of C3d with CR2, and the appearance of the B cell receptor-coreceptor complex, has been gained throughout evolution, as it is demonstrated by studying the electrostatic character of C3d from a variety of species using the concept of perturbation-resistant electrostatic hotspots [4]. Primitive species have only complement-mediated immunity, whereas more advanced species have both complement-mediated and antigen-antibody-mediated immunity. It is proposed that electrostatic hotspots in C3d have evolved to optimize its dual functionality, i.e. covalent attachment to pathogen surfaces and interaction with CR2.

The CR2-binding site in human C3d is multi-functional, also mediating electrostatic interactions with complement regulators, such as Factor H [5]. Such regulation is important to inhibit complement attack against host tissues. In addition, S. aureus bacterial proteins use the CR2-binding site of C3d to disrupt the function of the complement system, through electrostatically-driven binding [6, 7]. Moreover, the C3d (TED) domain of C3b is a site of electrostatic interactions with complement regulators Factor H, MCP, and DAF, as well as with viral proteins that mimic complement regulators to facilitate viral evasion of the complement system [8].

Compromised regulation of the complement system results to autoimmune and inflammatory diseases, such as lupus, rheumatoid arthritis, age-related macular degeneration, and rare kidney diseases. Given the fact that C3d is a biomarker of complement activation, C3d can be used as a diagnostic for early onset of complement-mediated diseases, prior to clinical diagnosis. A virtual screening study of 7 million chemical compounds for binding against C3d has led to the identification of 10 C3d ligands with micromolar affinities and fluorescence properties [9]. These compounds have the potential to become diagnostics for age-related macular degeneration, using in vivo fluorescence imaging. They also have the potential for dual therapeutic and diagnostic (theranostic) role, as well as for becoming drug carriers for targeted delivery at sites of local complement-originating inflammation.

[1] Zewde N, Gorham RD Jr, Dorado A, Morikis D (2016) Quantitative modeling of the alternative pathway of the complement system, PLOS ONE 11:e0152337.

[2] Mohan R, Gorham RD Jr, Morikis D (2015) A theoretical view of the C3d:CR2 binding controversy, Molecular Immunology 64:112-122.

[3] Mohan RR, Huber GA, Morikis D (2016) Electrostatic steering accelerates C3d:CR2 association, Journal of Physical Chemistry B, in press â?? available online.; DOI: 10.1021/acs.jpcb.6b02095.

[4] Kieslich CA, Morikis D (2012) The two sides of complement C3d: evolution of electrostatics in a link between innate and adaptive immunity, PLoS Computational Biology 8:e1002840.

[5] Harrison R, Gorham RD Jr, Morikis D (2015) Energetic evaluation of binding modes in the C3d and factor H (CCP 19-20) complex, Protein Science 24:789-802.

[6] Gorham RD Jr, Rodriguez W, Morikis D (2014) Molecular analysis of the interaction between staphylococcal virulence factor Sbi-IV and complement C3d, Biophysical Journal 106:1164-1173.

[7] Gorham RD Jr, Kieslich CA, Morikis D (2012) Complement inhibition by Staphylococcus aureus: electrostatics of C3d-EfbC and C3d-Ehp association, Cellular and Molecular Bioengineering 5:32-43. [8] Ojha H, Panwar HS, Gorham RD Jr, Morikis D, Sahu A (2014) Viral regulators of complement activation: structure, function and evolution, Molecular Immunology 61:89-99.

[9] Gorham RD Jr, Nuñez V, Lin J-H, Rooijakkers SHM, Vullev VI, Morikis D (2015) Discovery of small molecules for fluorescent detection of complement system activation product C3d, Journal of Medicinal Chemistry 58:9535-9545.