Chimeragenesis of distantly-related proteins by noncontiguous recombination

Abstract

We introduce a method for identifying elements of a protein structure that can be shuffled to make chimeric proteins from two or more homologous parents. Formulating recombination as a graph-partitioning problem allows us to identify noncontiguous segments of the sequence that should be inherited together in the progeny proteins. We demonstrate this noncontiguous recombination approach by constructing a chimera of β-glucosidases from two different kingdoms of life. Although the protein's alpha-beta barrel fold has no obvious subdomains for recombination, noncontiguous SCHEMA recombination generated a functional chimera that takes approximately half its structure from each parent. The X-ray crystal structure shows that the structural blocks that make up the chimera maintain the backbone conformations found in their respective parental structures. Although the chimera has lower β-glucosidase activity than the parent enzymes, the activity was easily recovered by directed evolution. This simple method, which does not rely on detailed atomic models, can be used to design chimeras that take structural, and functional, elements from distantly-related proteins.

Figure 1

Noncontiguous recombination. (a) One or more structures and a parental sequence alignment are used to identify contacts that are not conserved and can be disrupted on recombination (SCHEMA contacts). (b) Sequence elements that should be inherited together (blocks) are identified based using the SCHEMA contact map. Optimal blocks are often noncontiguous along the polypeptide chain but are contiguous on the three-dimensional structure. (c) The chimeras are reassembled using blocks from different parents. (d) The SCHEMA contact map can be reformulated as a graph, where nodes represent residues and edges represent SCHEMA contacts. (e) To design noncontiguous recombination chimera libraries, the graph is partitioned, with each residue assigned to a block. Partitions are chosen to minimize the edges between blocks. (f) Graph schematic of a chimeric protein.

Figure 2

β-Glucosidase noncontiguous chimera design chosen for construction. (a) Numbered sequence alignment of the eukaryotic (top) and prokaryotic (bottom) β-glucosidases. Conserved residues are in gray, the block of eukaryotic mutations are in red, and the block of prokaryotic mutations are in green. (b) The two-block design illustrated on the structure of the prokaryotic enzyme, TmBglA (2WBG.pdb).

Figure 3

The optimal noncontiguous design breaks far fewer contacts than random two-block partitions of the structure. (a) A histogram of the SCHEMA energies of 10,000 random two-block chimeragenesis designs. The SCHEMA energy of the optimized noncontiguous design is highlighted with a red arrow. (b) The SCHEMA contact map for the optimized noncontiguous two-block design. Most of the SCHEMA contacts are within the two blocks and thus are not disrupted on recombination. The numbering is based on the parent alignment, and SCHEMA contacts are shown in black. Red and green areas show the two blocks (For greater clarity, the conserved residues have been assigned to one of the two blocks based on structural proximity.)

Figure 4

Structural elements are conserved on recombination. (a) The structure of chimera NcrBgl (4GXP.pdb), bottom, is nearly identical to the assembled structure of its component blocks from TrBgl2 (3AHY.pdb) and TmBglA (2WBG.pdb), top. The eukaryotic TrBgl2 residues and the prokaryotic TmBglA residues are highlighted in red and green, respectively. (For greater clarity, the conserved residues have been assigned to one of the two blocks based on structural proximity.) (b) A structural alignment of TmBglA 2WBG.pdb and TrBgl2 3AHY.pdb (RMSD = 3.34 Å) shows significant variation between these two homologs. (c) An example of significant variations in loop regions. (d) Model of NcrBgl constructed simply by stitching together the parental blocks closely aligns with NcrBgl's actual structure (RMSD = 1.15 Å).

Figure 5

Directed evolution recovers the activity of NcrBgl to wild-type levels. Activity is measured in lysate with a 1-h assay on pNPG at 37°C and normalized relative to NcrBgl. The new mutations found at each round are listed (numbering based on the parental alignment). Five rounds of directed evolution increased the activity of NcrBgl almost 1000-fold.