Journal of Molecular Biology
Regular articleHighly divergent dihydrofolate reductases conserve complex folding mechanisms1
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 (IBP) 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.
Section snippets
Sequence and structural analysis
The structures of all DHFRs have striking similarity: structural alignments of hsDHFR, ecDHFR and lcDHFR (Figure 2(a)) indicated backbone RMSD values between 1.5 and 2.0 Å. The vertebrate enzymes are approximately 30 residues longer and result in the presence of external loops in the eukaryotic forms.14, 17 Despite the well-conserved backbones pairwise sequence identities are low: hs/ec, 29.2 %; ec/lc, 27.0 %; and lc/hs, 27.8 % (Figure 2(b)). Only ten residues are identical throughout the 78
Similarities and differences in folding mechanisms
The folding mechanisms for ec-, lc- and hsDHFR are fundamentally very similar, despite the low conservation of the amino acid sequence (∼30 % pairwise sequence identity). All three systems fold to the native state via parallel channels with two types of on-pathway kinetic intermediates. The deduced folding mechanisms for all three DHFRs are depicted in Scheme 1.Table 1 highlights the similarity of the unfolding and refolding relaxation times for the ecDHFR and hsDHFR homologs; the refolding
Materials
Ultrapure urea was purchased from ICN Biomedicals Inc. (Aurora, OH) and purified further using a mixed-bead, anion-exchange resin (AG™ 501-X8; Biorad). Folic acid, dihydrofolate, β-NADP+, methotrexate and recombinant human cyclophilin were purchased from Sigma. ANS and β-NADPH were obtained from Molecular Probes (Eugene, OR) and Amersham Life Sciences (Piscataway, NJ), respectively. All other reagents were of analytical grade. The concentrations of ligands were determined spectrophotometrically
Acknowledgements
We gratefully acknowledge the initial research on hsDHFR performed by Dr Jing Zhang. We thank Dr Jill A. Zitzewitz and B. Robert Simler for a critical reading of the manuscript, and Drs Roxana Ionescu and John O’Neill for fruitful discussions. This work was supported by National Science Foundation grants MCB-9604678 and MCB-0081076 to C.R.M. L.A.W. was supported, in part, by an NRF Postdoctoral Fellowship (South Africa).
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Edited by P. E. Wright