Why reinvent the wheel? Building new proteins based on ready-made parts

Abstract

We protein engineers are ambivalent about evolution: on the one hand, evolution inspires us with myriad examples of biomolecular binders, sensors, and catalysts; on the other hand, these examples are seldom well-adapted to the engineering tasks we have in mind. Protein engineers have therefore modified natural proteins by point substitutions and fragment exchanges in an effort to generate new functions. A counterpoint to such design efforts, which is being pursued now with greater success, is to completely eschew the starting materials provided by nature and to design new protein functions from scratch by using de novo molecular modeling and design. While important progress has been made in both directions, some areas of protein design are still beyond reach. To this end, we advocate a synthesis of these two strategies: by using design calculations to both recombine and optimize fragments from natural proteins, we can build stable and as of yet un-sampled structures, thereby granting access to an expanded repertoire of conformations and desired functions. We propose that future methods that combine phylogenetic analysis, structure and sequence bioinformatics, and atomistic modeling may well succeed where any one of these approaches has failed on its own.

Keywords: Rosetta; antibody; bioinformatics; evolution; protein design; β-propeller; β/α barrel.

Figure 1

Conserved catalytic machinery on widely different active‐site backbones (A) Two members of the amidohydrolase family: phosphotriesterase (PTE, magenta) and lactonase (PLL, cyan) have remarkably conserved core catalytic groups, comprising two metal ions (green spheres) and chelating residues (sticks). (B) Divergence of the active‐site loops leads to quite different binding sites of PTE (C) and PLL (D). Substrates shown as sticks and protein in surface. Molecular representations were generated using PyMol [The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.].

Figure 2

Examples of structural modularity in functionally versatile folds. A. β/α barrel structure (PDB ID: 1THF), with a quarter‐barrel highlighted in magenta. B. The five‐bladed β‐propeller structure (PDB ID: 1TL2), with a single blade subunit highlighted in magenta. C. Overlay of two antibodies, showing the conserved framework (gray) and divergent complementarity determining regions (CDRs). Molecular structures taken from the murine fluorescein‐binding antibody (PDB ID: 1X9Q, magenta) and an influenza hemagglutinin‐binding antibody from human (PDB ID: 3GBN, cyan).

Figure 3

A. Correlation of backbone segment conformations in antibodies with segment sequences (the conformations are colored according to their sequence profile). B. Essential non‐local stabilizing interactions of CDRs. Conformation of CDR H1 (magenta) of fluorescein binding antibody (PDB ID: 1X9Q) is supported by residues from adjacent framework and CDR regions, forming a complex network of nonlocal polar and hydrophobic interactions.

Figure 4

A. Conformation segmentation used in AbDesign. AbDesign segments the antibody structure in places of highest structure conservation among antibodies (the disulfide‐bonded cysteines, shown as sticks, and the stem positions of CDR3) to improve the potential of different conformation segments to be joined to form artificial combinations of backbones. The structure (PDB ID: 1X9Q) is color‐coded by conformation segments (red: CDRs L1&L2, green: CDR L3, blue: CDRs H1 and H2, yellow: CDR H3). Gray segments are not subjected to backbone design and are left as in the template antibody. B. Conformation segmentation used in design of TIM barrels. The basic modular unit, β − α−β, is shown in magenta on a phosphotriesterase (PTE, PDB ID: 1HZY). C. The stems, chosen as residues at the interface between β strands and α helices that are well‐aligned across the family, are shown as cyan sticks.