Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

Abstract

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.

Figure 1

Scheme of mDHFR fragmentation and of the DHFR fragment complementation assay. The homodimerizing GCN4 leucine zipper (Zipper), and mDHFR fragments (1, 2, and 3) are illustrated. The labels for the constructs are used to identify both the DNA constructs and the proteins expressed from these constructs.

Figure 2

Structure of hDHFR (32) showing fragments F[1,2] (blue-cyan) and F[3] (yellow) bound NADPH (violet) and methotrexate-γ-tetrazole (gold). (A) Natural and fragment N and C termini (N, C, and N′, C′, respectively) are indicated by blue or red circles. Arrows indicate location of fusion to oligomerizing proteins. Position of Ile-114 and contact residues in F[1,2] (Ile-51 and Leu-93) are indicated. (B) Ninety degree vertical axis rotation of view in A showing positions of methotrexate resistance mutations.

Figure 3

(A) E. coli survival assay on minimal medium plates. Control, left side of the plate: E. coli harboring pQE-30 (no insert); right side: E. coli expressing full-length mDHFR. (A, I) Left half of each plate: transformation with Z-F[1,2]; right half of each plate: transformation with Z-F[3]. (A, II) Cotransformation with Z-F[1,2] and Z-F[3]. (A, III) Cotransformation with constructs control-F[1,2] and Z-F[3]. All plates contain 0.5 μg/ml trimethoprim and plates on the right side of I-III contain 1 mM IPTG. (B) E. coli survival assay using destabilizing DHFR mutants. (B, I) Cotransformation with Z-F[1,2] and Z-F[3:Ile-114 → Val]. (B, II) Cotransformation with Z-F[1,2] and Z-F[3:Ile-114 → Ala]. (Inset) A 5-fold enlargement of the right-side plate. (B, III) Cotransformation with Z-F[1,2] and Z-F[3:Ile-114 → Gly]. All plates contain 0.5 μg/ml trimethoprim and plates on the right side contain 1 mM IPTG.

Figure 4

E. coli survival assay as in Fig. 3 Ras and Raf (A). Cotransformation with raf-F[1,2] alone (I); ras-F[3] alone (II); raf-F[1,2] and ras-F[3] (III); raf-F[1,2] and zip-F[3] (IV), and zip-F[1,2] and ras-F[3] (V). (B) FKBP and TOR2. Cotransformation with FKBP-F[1,2] alone (I); TOR2-F[3] alone (II); FKBP-F[1,2] and TOR2-F[3] (III); vector alone (IV); zip-F[1,2] and TOR2-F[3] (V). (C) As in B, in the presence of 1 μM rapamycin.