Routes to covalent catalysis by reactive selection for nascent protein nucleophiles

Abstract

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.

Figure 1

Alignment of covalent and non-covalent binding clones. The conserved amino acid residues of the heavy chain are highlighted in yellow and those of light chain in green. CDR regions are enclosed in boxes. Sequences of covalent binding clones (designated as A.nn), are shown in bold font.

Figure 2

Structural alignment of A.5 and A.17 VH and VL sequences. The CDR regions are indicated in colored highlight. The tryptic peptides spanning the residues modified by 1 are enclosed in the blue box. Reactive tyrosine residues are shown in red font. Residues of VH and VL and CDRs are identified according to the Kabat numbering system.

Figure 3

(A) Covalent reactivity of A.17 and A.5 and their mutants. Concentrations of scFvs were normalized as confirmed by comparable staining in anti-c-Myc blot (lower panel). (B) Binding efficiency of clones selected after the third round, including reactive clones A.5, A.17 and their non-reactive mutants A.5Y32F and A.17Y36F. Non-selected clone (N) and anti-thyroglobulin scFv (anti-Thyr) were included as negative controls. Binding was determined as described in Methods.

Figure 4

Reaction of A.17 with alternative substrates. A.17 was incubated for 1 hour at 37°C with 5 mM each of AEBSF (lane 1), 3 (lane 2), or 4 (lane 3), or 100 μM of 5 (lane 4), 6 (lane 5), or 2 (lane 6), or PBS buffer alone (lane 7). All samples were then incubated for 1 hour at 37°C with 100 μM of 1 and analyzed by western blot as in Figure 3A.

Chart 1

Reactive phosphonates used for selection and assay of scFv.