Highly divergent dihydrofolate reductases conserve complex folding mechanisms¹

Abstract

To test the hypothesis that protein folding mechanisms are better conserved than amino acid sequences, the mechanisms for dihydrofolate reductases (DHFR) from human (hs), Escherichia coli (ec) and Lactobacillus casei (lc) were elucidated and compared using intrinsic Trp fluorescence and fluorescence-detected 8-anilino-1-naphthalenesulfonate (ANS) binding. The development of the native state was monitored using either methotrexate (absorbance at 380 nm) or NADPH (extrinsic fluorescence) binding. All three homologs displayed complex unfolding and refolding kinetic mechanisms that involved partially folded states and multiple energy barriers. Although the pairwise sequence identities are less than 30 %, folding to the native state occurs via parallel folding channels and involves two types of on-pathway kinetic intermediates for all three homologs. The first ensemble of kinetic intermediates, detected within a few milliseconds, has significant secondary structure and exposed hydrophobic cores. The second ensemble is obligatory and has native-like side-chain packing in a hydrophobic core; however, these intermediates are unable to bind active-site ligands. The formation of the ensemble of native states occurs via three channels for hsDHFR, and four channels for lcDHFR and ecDHFR. The binding of active-site ligands (methotrexate and NADPH) accompanies the rate-limiting formation of the native ensemble. The conservation of the fast, intermediate and slow-folding events for this complex α/β motif provides convincing evidence for the hypothesis that evolutionarily related proteins achieve the same fold via similar pathways.

Introduction

The tertiary structures of homologous proteins, i.e. those with similar biological functions that have evolved from a common ancestral gene, are known to be better conserved than their amino acid sequences.1 Reasoning that the conservation of the product of a folding reaction might also require the conservation of key folding intermediates and/or transition states, it has been proposed that folding mechanisms of proteins have been conserved during evolution.2, 3

The validity of this hypothesis has been supported by a number of experimental studies on small (< 100 residues), single-domain proteins that have relatively high levels of sequence similarity (for a review, see Gunasekaran et al.4 and references therein). For the proteins that fold via a simple two-state mechanism, e.g. the family of all-β SH3 domains,5, 6, 7, 8 the properties of the transition state and its position on the reaction coordinate diagram were highly conserved. Even simplifying the amino acid code of the SH3 domains to five letters does not cause a dramatic change in the rate of folding or in the position of the transition state.9 Further, molecular dynamic unfolding and ab initio refolding simulations10 for the SH3 domains were consistent with the experimental findings.

Although a few studies comparing the complex folding mechanisms of larger and/or multi-domain proteins have been published (see Stackhouse et al.11 and Gunasekaran et al.4) it is not yet clear whether rate-limiting steps that occur after the development of chain topology are also conserved. The family of dihydrofolate reductases (DHFRs, EC. 1.5.1.3) is an ideal protein system with which the conservation of folding pathways hypothesis can be tested. DHFRs, which function in vivo by maintaining the pool of dihydrofolate and its derivatives that are essential for the biosynthesis of purines, thymidylates and several amino acids (for reviews, see Blakely12, 13), span the three domains (archae, prokaryote and eukaryote) of life. All representatives whose structures have been solved thus far have an α/β/α topology where the mixed eight-stranded β sheet is flanked on either side by two α helices.14, 15, 16, 17, 18, 19 The complex, doubly wound β-sheet topology is composed of two structurally distinct domains; an adenosine-binding domain (ABD) common to all nucleotide-binding proteins, and a loop subdomain (Figure 1; and see Sawaya et al.17).

The thermodynamic properties and kinetic folding mechanism of Escherichia coli (ec) DHFR have been studied in detail, using a wide variety of biophysical techniques.20, 21, 22, 23, 24, 25, 26, 27, 28 The proposed kinetic folding mechanism for ecDHFR is complex. The first detectable step in folding involves a rapid collapse (<5 ms) of the urea-denatured state to form a burst phase species (I_BP) that has significant secondary structure22 and an exposed hydrophobic core.24 The results of pulse-labeling hydrogen exchange experiments in combination with NMR25 have been interpreted to imply that this core involves two clusters of non-polar side-chains that stabilize the native β-sheet topology in 60 % of the population. Mutational analysis28 has demonstrated stereo-specific packing in these non-polar side-chains. The rapid establishment of the β-sheet topology is followed by the formation of a set of hyperfluorescent intermediate states (I₁-I₄; τ = 200 ms) that convert to the native states (N₁-N₄) via four parallel folding channels (relaxation times in the range of 1-100 seconds). All of the native states bind the competitive inhibitor methotrexate (MTX), but only one native state (N₂) has the ability to bind the cofactor, NADP⁺. The richness of this kinetic mechanism, including two distinct folding reactions that follow the chain organization step, provides the basis for a stringent test of the conservation of folding mechanisms hypothesis.

The thermodynamic properties and folding kinetics of human (hs) DHFR and Lactobacillus casei (lc) DHFR were elucidated and compared with those for ecDHFR. Although the pairwise sequence identities are less than 30 %, the comparison demonstrated that the complex folding mechanism is conserved throughout the family of DHFRs.