Partially folded states of staphylococcal nuclease highlight the conserved structural hierarchy of OB-fold proteins.

The concept of a protein fold as a recurrent unit of structure is central to modern structural biology. One of the goals of The Protein Structure Initiative is to determine representative examples of all folds (1), estimated to number between 1000 (2) and 10 000 (1, 3, 4). The difficulty of protein structure prediction targets and the approaches for modeling unknown structures are often predicated on whether the protein is likely to have a novel fold (5). Inferences about evolutionary relationships are often made on the basis of protein folds, because structure can be conserved even when sequences have diverged to levels where sequence homology is no longer detectable (6). While the concept of a protein fold is generally accepted, the classification of folds in practice remains somewhat subjective and ambiguous. The OB-fold1 (Figure 1) was originally described as an oligonucleotide/oligosaccharide-binding fold (7) and consists of a five-stranded β-barrel structure. It is represented well in both sequence and structure databases (3, 8–10). The October 2006 release of the SCOP database (11) classifies some 95 protein domains as OB-fold structures. These are grouped into 27 function families with activities ranging from molybdate-binding proteins to RNA chaperones. These families are further grouped into 10 superfamilies that are hypothesized to have a common evolutionary origin (11). Figure 1 Structural variability in OB-fold proteins. (A) Proteins currently assigned to the OB-fold in the SCOP database (69) range from a three-stranded β-meander (PDB entry 1RL2) to a 10-stranded β-sheet thought to correspond to a domain-swapped ... Representative structures classified as OB-fold domains in the SCOP database are shown in Figure 1A. The secondary structures range from 3 to 10 strands of β-sheet. Many of these proteins have additional α-helices that are not conserved. It has been duly noted that structure similarity spans a continuum, much like sequence similarity (3). As such, the classification of protein structures according to discrete “folds” may be somewhat artificial, particularly if the point of the classification is to infer functional relationships. Another motivation for the classification of proteins into fold families is to discern evolutionary relationships (1). Here, care must be exercised to distinguish convergent from divergent evolution (12). Structures related through divergent evolution are derived from a shared immediate ancestor, whereas in convergent evolution, similar structural features arise independently to satisfy a functional role. Our working hypothesis is that information about protein folding can help distinguish evolutionary relationships among structures, much in the same way that protein structures can reveal homology not apparent from a comparison of protein sequences. To this end, our lab has carried out comparative folding studies of three proteins from the nucleotide-binding branch of the OB-fold family (Figure 1B): CspA (cold shock protein A), LysN (anticodon binding domain of lysyl tRNA synthetase), and SN (staphylococcal nuclease). CspA is a mRNA chaperone that binds mRNA in such a way that it facilitates translation when Escherichia coli are grown at low temperatures (13). LysN recognizes the anticodon loop of lysyl tRNA (14). SN is a phosphodiesterase secreted by Staphylococcus aureus that hydrolyzes DNA or RNA to 3′-mononucleotides in the presence of Ca2+ (15). The three proteins bind oligonucleotides roughly on the same location of their β-barrels, with most of the polar residues involved in substrate binding coming from loops L12, L34, and L45 (7). The conservation of structure and ligand recognition motifs makes the OB-fold an excellent model system for determining if similarity also extends to folding mechanisms. We initially looked at the proteins using mutagenesis and found that the OB-fold has the properties of an independent folding module (16, 17). To obtain more complete information, we extended our comparative studies to the native-state hydrogen exchange (NSHX) method (18, 19). Hydrogen exchange (HX) is a powerful tool for studying the stabilities and dynamics of proteins. Exposed amide protons exchange with solvent according to intrinsic rates that can be calculated from the solution pH, temperature, and amino acid sequence (20, 21). Exchange can be slowed by structure, and the ratio of observed to intrinsic rates gives site-specific information about the stability of structure (20). Protected amide protons can exchange by two mechanisms: localized noncooperative breathing motions or unfolding of part or the entire structure. The mechanisms can be distinguished from the denaturant dependence of HX, which forms the basis of the NSHX method (19, 20). Localized breathing motions expose little new surface area to solvent and are independent of denaturant concentration. By contrast, cooperative partial or complete unfolding of the protein exposes a large new surface area to solvent, and plots of exchange rates versus denaturant concentration have non-zero slopes (mHX values). The NSHX experiments are conducted over a range of denaturant concentrations where the native state always predominates (>99% N state). Exchange occurs through transiently formed high-energy states that become more populated as the denaturant concentration increases (20, 22). The dependence of the data on denaturant concentration provides information about the stability of the native structure in the absence of denaturant ( ΔGunfo) and about the cooperativity of unfolding (mHX). We previously used the NSHX experiment to characterize the free energy landscape of CspA and LysN. Here we bring these studies to completion with an investigation of SN. The results show that the structurally conserved β-barrel portion of the structure is more stable to unfolding and more cooperatively stabilized than the nonconserved elements of structure. The hierarchy of structural stability in SN is similar to that previously defined for LysN and CspA. In addition to the NSHX experiments, we obtained data at pH 10.2 where exchange is in the EX1 limit and determined the rates with which “open” exchange-susceptible conformations are generated for the subset of slowest exchanging amide protons (22, 23). These experiments indicate that the structurally conserved OB-fold core, which has the greatest stability to unfolding, also has the slowest unfolding rates.