De Novo Protein Design of Agonists and Antagonists of the C3a Receptor: Theoretical Predictions and Experimental Validation

Protein design, also known as the inverse folding problem, seeks the amino acid sequence that will fold into a given 3-dimensional template. The protein design problem exhibits degeneracy due to the fact that many amino acid sequences fold into a given template. It is therefore important to examine all the possible sequences for a given template and rank them based upon specific properties that are being designed (activity, specificity, etc.).

A de novo design framework with a ranking metric based on fold specificities was applied to the design of C3a receptor (C3aR) agonists and antagonists. The design is based upon the structure of C3a, which activates C3aR. C3a is a 77-residue peptide that mediates the pro-inflammatory activities in the human complement system and possibly has opposing immunological roles in some cellular systems (34). Improper activation of the complement system can cause tissue injury in various pathological conditions and contributes to several immune diseases, including stroke, heart attack, Alzheimer's disease, asthma, rheumatoid arthritis, and rejection of xenotransplantation. The crystal structure resolved by Huber et al. [1], as well as flexible template structures generated by MD simulations with explicit solvation via water molecules were employed as the design template.

The framework consists of two stages: a sequence selection stage and a validation stage. The sequence selection stage produces a rank-ordered list of amino acid sequences with the lowest energies by solving an integer programming sequence selection model [2]. The sequence selection model incorporates backbone flexibility into the design process by utilizing the set of structures obtained from the MD simulations with implicit solvation and with explicit water molecules. In doing so, the pairwise energy between two residues can take on a range of values depending upon the range of distances obtained from the structures.

The second stage re-ranks the sequences from stage one using either a fold specificity or an approximate binding affinity [3-4]. Since structural information of the C3a:C3aR complex was unknown, only fold specificity calculations could be employed. Fold specificity measures how likely a given sequence will fold into the design template structure [5]. Thus the design was driven by the hypothesis that structure implies function, and novel sequences of C3a that adopt the C3a fold are potential candidates for C3aR agonists or antagonists.

Hundreds of sequences were generated and ranked using the de novo protein design framework. The top seven sequences were selected for synthesis and experimental validation. Of the seven designed peptides tested, two were prominent agonists while two others were prominent antagonists. The agonists showed a up to a five-fold improvement in EC50 compared with the previously discovered "superagonist" by Ember et al. [6], with EC50s of 33.4 and 66.16 nM. The antagonists were also very potent, with IC50s of 26.13 and 63.04 nM.

This work highlights the success of the protein design framework. In this case, structural information on the binding site was unknown, so the design was driven by fold specificities. Since structure often implies function, this is a good metric to use when structural binding data is unavailable.

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[3] Bellows, M. L., H. K. Fung, C. A. Floudas, A. Lopez de Victoria, and D. Morikis, 2010. New Compstatin Variants Through Two De Novo Protein Design Frameworks. Biophys J 98:2337–2346.

[4] Bellows, M. L., M. S. Taylor, P. A. Cole, L. Shen, R. F. Siliciano, H. K. Fung, and C. A. Floudas, 2010. Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework. Biophys J 99:3445–3453.

[5] Fung, H. K., C. A. Floudas, M. S. Taylor, L. Zhang, and D. Morikis, 2008. Toward Full-Sequence De Novo Protein Design with Flexible Templates for Human Beta-Defensin-2. Biophys. J. 94:584–599.

[6] Ember, J., N. Johansen, and T. Hugli, 1991. Designing Synthetic Superagonists of C3a Anaphylatoxin. Biochem. 30:3603–3612.