Designing artificial enzymes by intuition and computation

Abstract

The rational design of artificial enzymes, either by applying physico-chemical intuition of protein structure and function or with the aid of computational methods, is a promising area of research with the potential to tremendously impact medicine, industrial chemistry and energy production. Designed proteins also provide a powerful platform for dissecting enzyme mechanisms of natural systems. Artificial enzymes have come a long way from simple α-helical peptide catalysts to proteins that facilitate multistep chemical reactions designed by state-of-the-art computational methods. Looking forward, we examine strategies employed by natural enzymes that could be used to improve the speed and selectivity of artificial catalysts.

Figure 1. From Sequence to Structure to Function

(A) A repeating seven residue pattern of nonpolar (●) and polar (○) residues will create a hydrophobic surface on one face of the helix. These surfaces can drive associations of helices, forming a hydrophobic core. (B) The oxaldie enzymatic peptides make dual use of this repeating pattern, creating one hydrophobic, leucine rich face and one cationic, lysine rich face. Spatial clustering of lysines lowers their pK_a and provides a surface for the negatively charged oxaloacetate substrate to bind. L = leucine (grey), K = lysine (red), A = alanine(white).

Figure 2. Helical designs with tertiary structure

Using short connecting loops, multi-helical structures provide the potential for greater chemical diversity at the active site, a binding surface for defining substrate specificity and a core for controlling the microenvironment of key catalytic residues. Two examples are the HN1 ribonuclease with an active site of two histidines and four arginines (model structure), and α₃W , a three helix bundle which tunes the reduction potential of a tryptophan radical in the bundle core.

Figure 3. Step-wise oxygen binding to a binary-patterned four helix bundle

In the HP-7 maquette, heme binding requires rotation of a-helices to present histidines (green) in the proper geometry. This forces the unfavorable burial of glutamates (red triangles) inside the nonpolar protein core. Release of one of the axial histidines allows the glutamates to interact with solvent and provides an open coordination site on the heme for oxygen binding. (Figure adapted from reference )

Figure 4. Retrostructural analysis and design of a dinuclear metalloprotein

The DF series of designed metalloenzymes were built from structural analysis of dinuclear metal sites in proteins such as ribonucleotide reductase . The two metals, two histidines and four glutamates that formed the active site could be described by two half-sites related by a C2 symmetry axis. The same symmetry was found locally in the natural metalloenzymes and was used in the de novo design of a helix-loop-helix dimer, DF-1.

Figure 5. Design versus Structure of a Zn2+ Binding MBP

(A) Using the computer program DEZYMER, four substitutions were designed into closed form of the Maltose Binding Protein (MBP) which conferred affinity for zinc. Mutations (in yellow) were in the maltose binding site (white spheres). (B) Crystal structures of the MBP variant demonstrated binding was in fact to a conformation closer to the open form, only involving half of the residues and an additional aspartate ligand (green). A = alanine, R = arginine, Y = tyrosine, W = tryptophan, E = glutamate, D = aspartate, H = histidine.

Figure 6. Assembly line for the ROSETTA Enzymes

(A) A reaction motif is sketched, outlining key intermediates, general acid base ligands, and strategies for modulating catalytic residue pK_as, (B) QM calculations are used to optimize the geometry of a transition state model including truncated active site residues. (C) Elaboration of catalytic residue rotamers creates an ensemble of active sites. (D) These are matched to complementary surfaces on a family of target protein scaffolds. (E) Promising designs are synthesized and characterized for activity. (Figure adapted from reference ).

Figure 7. Millisecond time-scale motions in adenylate-cyclase

Adenylate cyclase maintains intracellular concentrations of adenylate nucleotides by converting an ATP and an AMP into two ADP molecules. Binding and catalysis requires a significant conformational rearrangement.^,