Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops

Abstract

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 beta-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two beta-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first beta-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final beta-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.

Figure 1.

Structure of TEM-1 β-lactamase. The essential nucleophilic residue, Ser70, is shown in yellow; L₁, L₂, and L3, the loops in which random peptides were inserted, are shown in green, blue, and orange, respectively.

Figure 2.

Characteristics of combination library fd-blaA-L₁ ^RL₂ ^CxCL₃ ⁶. Twenty-eight clones from the total library of 2.9 × 10⁸ clones and 120 from the active library of 1.35 × 10⁷ clones were sequenced. The color code is as follows: (1) two complete inserts in L2 and L3 (2.8 × 10⁸ clones in the total library, 10⁶ clones in the active library); (2) a complete insert in L2 and an insert smaller than six residues in L3; (3) a complete insert in L2 and a mutated residue in L3; (4) a complete insert in L2 and a nonmutated T271 in L3. Residues in L1 are mutated. The inner circle is the fraction of the library corresponding to the active clones. It is magnified for easier observation of the various types of clones in this portion of the combination library.

Figure 3.

Relative amino acid occurrences in the three positions of loop L1 (according to the Ambler numbering; residue 239 is missing in TEM-1 β-lactamase). The relative occurrence was calculated as the ratio of the observed occurrence (x _obs) to that expected from the total number of observations (n = number of clones sequenced), given the number of codons coding for a particular residue (x _exp).

Figure 4.

Relative amino acid occurrences in the extended loops L2 (A) and L3 (B). The relative occurrence was calculated as the ratio of the observed occurrence (x _obs) to that expected from the total number of observations (n = number of clones sequenced), given the number of codons coding for a particular residue (x _exp). The amino acids are arranged in order of decreasing propensity of presence in Ω-loops of high accessibility computed from a large database (Pal and Dasgupta 2003) and are shown as three groups of high, medium, and low mean Ω-loop preference, indicated by white, gray, and black bars. The significance of the deviation of the x _obs/x _exp ratio is shown as significant (*), highly significant (**), or very highly significant (***), with the probability of a chance observation being 5%, 1%, and 0.1%, respectively (see Materials and Methods).