Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase fromE. coli: Fragment complementation and circular permutation reveal stable, alternatively folded forms

Abstract

The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods. Cleavage of the backbone after position 86 resulted in two fragments, {1-86} and {87-159}, each of which are poorly structured and enzymatically inactive. When combined in a 1 : 1 molar ratio, however, the fragments formed a high-affinity (K a ‫ס‬ 2.6 × 10 7 M −1) complex that displays a weakly cooperative urea-induced unfolding transition at micromolar concentrations. The retention of about 15% of the enzymatic activity of full-length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild-type counterpart. In contrast, a circularly permuted form with its N-terminus at position 86 has similar overall stability to full-length DHFR, about 50% of its activity, substantial secondary structure, altered side-chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild-type protein. After addition of ligand or the tightbinding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more nativelike secondary and tertiary structures. These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein-ligand interactions in stabilizing the active site architecture of DHFR.

Folding pathway of a multidomain protein depends on its topology of domain connectivity

“…At the characteristic times of t = τ A , τ B , and 3:0 × 10 6 t 0 , the distributions of positions of MC trajectories on the 2D space of ðM DLD ; M ABD Þ are shown in Fig.5 B-D. By comparing these results with the free-energy surface in Fig. 4A, no population of the simulated trajectories can be found that shifted toward I α , and the simulated DHFR folding follows a single sequential pathway of Path ABD , which is also consistent with experimental observations (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). At the later phase of folding, the internal friction should begin to increase the timescale for the folding process (30)(31)(32).…”

“…To analyze the effects of changing the topology, circular permutants of DHFR (cp-DHFR) have been experimentally investigated (22,28,29). In a circular permutant that was obtained by cutting the chain between residues 38 and 39 and connecting the N and C termini, both ABD and DLD comprise continuous parts of the chain, so that the topological complexity of wild-type DHFR, or the discontinuity in DLD, is resolved.…”

“…Because DLD does not include a single contiguous region of the chain, but rather includes separate N-and C-terminal parts, the structural ordering of DLD can be correlated with the structural ordering of ABD. As a model protein, DHFR has been intensively investigated (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), which has resulted in a picture that DHFR folds along the following pathway:…”

Circular Permutation of 5-Aminolevulinate Synthase

“…Certainly, the apo forms of ALAS and permuted variants were unstable and precipitated under our experimental conditions. The idea that determinants of protein structure and stability arise as a response to (natural) ligands has been previously advanced (52,57); indeed, the PLP cofactor contributes to the structural stabilization of several PLP-dependent enzymes, although its direct relevance to their folding mechanism varies (58 -60). Accordingly, we postulate that interdomain interactions stabilize the dimeric interface, where the PLP cofactor is cradled and the active site resides.…”

A simple theoretical model goes a long way in explaining complex behavior in protein folding

“…The role of proline isomerization was also included, which is important to describe the multiphasic kinetics often observed in multidomain protein folding. Armed with an extended WSME model, they tackled the analysis of the folding reaction of dihydrofolate reductase (DFHR), a two-domain protein previously studied experimentally by Matthews and coworkers (18). The experiments reveal a remarkably complex process with up to seven kinetic phases with timescales spanning over 6 orders of magnitude that reflect the formation of early intermediates combined with the slow isomerization of 10 proline residues.…”