A computational method for design of connected catalytic networks in proteins

Abstract

Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side-chain functional groups which maximize transition-state stabilization, and then searching for sites in protein scaffolds where the specified side-chain-transition-state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example, proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen-bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw-like two-dimensional representation of the transition state with hydrogen-bond donors, acceptors, and covalent interaction sites indicated, and all placements of side-chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen-bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully-connected active sites in protein scaffolds. The method generates many fully-connected active site solutions for a set of model reactions that are promising starting points for the design of fully-preorganized enzyme catalysts.

Keywords: biocatalysis; computational modeling; enzyme design; enzyme mechanism; protein design.

Figure 1

Comparison of the RA95.0 and RA95.5‐8F active sites suggests the importance of catalytic residue connectivity. (a) A two dimensional representation of the RA95.0 theozyme, which consists of Lys210, Glu53, plus a water molecule. (b) Directed evolution of RA95.0 yielded the 200,000‐fold more active RA95.5‐8F, whose catalytic groups include a repositioned lysine, Lys83, along with Tyr180, Tyr51, and Asn110. The three‐dimensional representation of the crystallographic coordinates of RA95.5‐8F are shown in (c) in the context of the complete scaffold, with the networked residues shown in magenta sticks, the ligand in cyan sticks, and hydrogen bonds indicated with black, dashed lines. Analysis of the crystal structure reveals an extensive network of hydrogen bonds between the catalytic side chains, preorganizing the active site. Three‐dimensional realizations of the RA95.5‐8F‐inspired active site generated by HBNetGen in three different protein scaffolds: (d) a lipid transfer protein (scaffold PDB accession code 1bwo); (e) a thymidine kinase (1w4r); and (f) an FKBP‐like domain (1c9h)

Figure 2

Generation of HBNetGen three‐dimensional (3D) active sites from two‐dimensional (2D) ChemDraw wiring diagrams. Eight candidate networks surrounding ligands were selected from the PDB: (a) (4R,5R)‐3‐amino‐4,5‐di‐ hydroxy‐cyclohexene‐1‐carboxylate; (b) 1,3‐dihydroxyacetone‐phosphate; (c) arginine; (d) {1‐[(3‐hydroxy‐methyl‐5‐phosphonooxy‐methyl‐pyridin‐4‐ylmethyl)‐amino]‐ethyl}‐phosphonic acid; (e) 3‐O‐ɑ‐D‐mannopyranosyl‐ɑ‐D‐mannopyranose; (f) maltose; (g) the open form of penicillin G; and (h) the retro‐aldol intermediate found in RA95.5‐8F. Each row shows: (left) a 2D representation of the interactions that form a complete network; (middle) the lowest‐energy network configuration at the functional‐group level, with the total number of network configurations indicated below; and (right) a full side‐chain representation of a network configuration, with total number of full side‐chain realizations for the network configuration (dependent on the number of rotatable bonds for the constituent side chains) indicated below. The total number of full side‐chain 3D realizations of the 2D connected reaction schematic on the left is the production of the numbers in the second and third columns: (number of 3D placements of functional groups) × (number of sidechain placements per functional group placement)