(111c) De Novo Protein Design With Flexible Backbone Templates And Its Application To The Redesign Of Complement 3a

De novo peptide or protein design starts with a flexible three-dimensional protein backbone and involves the search for all amino acid sequences that will fold into such a template ([1]-[3]) and exhibit higher stability and/or higher activity. It is of paramount importance to designing improved inhibitors, engineering proteins with higher stability, conferring novel properties to catalytic sites of enzymes, and speeding up the drug discovery process [5].

Our current de novo protein design framework consists of two stages: (i) in silico sequence selection, and (ii) fold validation. Recently, we have devised two new sequence selection models that handle explicitly true protein backbone flexibility [6], as exhibited by multiple X-ray or NMR structures. One applied the single structure algorithm on a weighted average of the multiple structures, and the other employed binary distance bin variables to account for each of the structures, but imposed constraints on C^α position pairs (i,k) and (k,p) to eliminate solutions which would otherwise suggest physically impossible structures [6]. Both models were developed based on our discovery of an efficient way for linearizing the sequence selection model for single protein structure [6]. The models treat true backbone flexibility explicitly because they consider protein conformations of all possible combinations of continuous dihedral angle and C^α-C^α distance values between bounds determined by the X-ray or solution structures. As far as fold validation is concerned, we are applying a highly computationally efficient method, CYANA, on a full-atomistic scale driven by the AMBER forcefield, together with a local energy minimization package TINKER, for calculating fold specificities of the new sequences from the stage one models.

In this work, the framework is applied to the design of Complement 3a (C3a), a 77-residue cleavage component of Complement 3 that is generated during the complement activation cascade. De novo design of C3a was based upon both the X-ray crystal structure elucidated by Huber et al. [7] and our own structures of the protein generated using molecular dynamics simulation with both generalized Born implicit solvation and explicit water molecules [8]. The design was aimed at obtaining an agonist that binds to the C3aR receptor better than C3a. In view of the multiple structures corresponding to the design template for C3a, the novel formulations using both a weighted average structure and binary distance bin variables were employed in the design work. Several sequences predicted for the agonist were confirmed experimentally to be able to compete with Ember et al.'s superagonist [8].

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[8]- J. A. Ember and N. L. Johansen and T. E. Hugli. "Designing Synthetic Superagonists of C3a Anaphylatoxin." Biochemistry 30 (1991): 3603-3612.