Functional evolution of two subtly different (similar) folds
© Agrawal and Kishan; licensee BioMed Central Ltd. 2001
Received: 27 August 2001
Accepted: 21 December 2001
Published: 21 December 2001
The function of proteins is a direct consequence of their three-dimensional structure. The structural classification of proteins describes the ways of folding patterns all proteins could adopt. Although, the protein folds were described in many ways the functional properties of individual folds were not studied.
We have analyzed two β-barrel folds generally adopted by small proteins to be looking similar but have different topology. On the basis of the topology they could be divided into two different folds named SH3-fold and OB-fold. There was no sequence homology between any of the proteins considered. The sequence diversity and loop variability was found to be important for various binding functions.
The function of Oligonucleotide/oligosaccharide-binding (OB) fold proteins was restricted to either DNA/RNA binding or sugar binding whereas the Src homology 3 (SH3) domain like proteins bind to a variety of ligands through loop modulations. A question was raised whether the evolution of these two folds was through DNA shuffling.
The analysis of protein structures as a group in generating and retrieving information is useful in various ways. The structural bioinformatics analysis of protein data bank (PDB)  is useful in identifying protein folds [2, 3] and identification of unknown protein functions. The analysis of some of the folds illustrated the packing arrangement of the secondary structural elements and features of various non-bonding interactions prevailed in these folds. This in turn helps in identifying active site residues of proteins of unknown functions. For example, the TIM-barrel fold, which is the most frequently observed fold has majority of members as enzymes and the active-site residues are situated on the loops connecting the β-strands to helices or at the C-terminal end of the parallel β-strands of the barrel . Therefore, for any enzyme having a Tim-barrel fold there is a possibility that the active site may be present at the same position consensus with other Tim-barrel fold enzymes.
Results and discussion
Search for SH3-fold and SH3 like folded proteins over various fold classification servers and manual literature search yielded a large number of protein domains. Some of the domains exist as individual proteins and some were part of a multi-domain protein. After superposing the protein domains on each other and through analysis for a common fold architecture we identified two folds, which are common in architecture but differ in topology. Here architecture is defined as immediate apparent similarity in fold irrespective of connectivity and topology is defined as the actual way the secondary structural elements are connected and come together to form a fold. One of the folds is known as OB-fold  and the other is SH3-fold. There are at least 30 proteins/domains classified as adopting these two folds [6–10] and the list is increasing. Although, there are more proteins/domains, which could be classified into one of the two folds, they were not included due to too many deviations from a consensus ensemble of structures.
To our surprise we observed that, while OB-fold always binds to either oligonucleotides or oligosaccharides, SH3-fold binds to a wide spectrum of ligands like DNA/RNA (Ribosomal protein L2 , Sso7d  and HIV Integrase DNA binding domain ), peptides (SH3 domains ) and folate (dihydrofolate reductase ). Although, few enzymes have SH3-folded domains as part of the enzyme, they stabilize the catalytic domain for optimal function (nitrile hydratase ) or stabilize the incoming ligand (ferridoxin:thioredoxin reductase ).
From figure 5 it is clear that the major difference between the two folds is the insertion/deletion of a β-strand, apart from the omega helix in OB-fold. Since the ligand-binding region in both folds is also similar, one could wonder whether these two folds were evolved from a common ancestor. If so, is it a function-driven protein evolution as argued by Fetrow and Godzik ? There are both negative as well as positive indicators to support this possibility. The fact that all the proteins considered in this study were not grouped into the same superfamily in the SCOP database  indicates that these two folds are not homologous or remotely homologous. The very low sequence homology and classification into different folds in SCOP suggests that they may not be analogous also. However, a simple concept of DNA shuffling, first worked out by Stemmer  and later demonstrated by many others, showed that new proteins and folds could be evolved through random fragmentation and reassembly [25–27]. On similar lines, SH3-fold and OB-fold could possibly be evolved from a common ancestor or evolved one from the other, through shuffling of small DNA segments over a large time-scale. Although there is no direct evidence to prove that these two folds are evolved from each other, directed-evolution experiments as demonstrated by Stemmer  may be useful to prove or disprove this hypothesis.
The common fold characteristics of both OB-fold and SH3-fold have diversified loops in sequence as well as in length. This feature prompts us to assume that these two folds could be used as a basic fold in designing new proteins with tailored functions. The designing of a chimeric protein with the basic fold of five strands from one protein and loops from another protein with appropriate mutations could be a starting point to test this hypothesis.
Materials and methods
The β-barrel proteins used for the analysis under SH3-fold were SH3 domain of chicken brain spectrin (1SHG), CcdB a topoisomearse poison from E. coli (4VUB), dihydrofolate reductase (1VIE), diphtheria toxin (1BYM), N-terminal domain of eucaryotic translation initiation factor 5a (1EIF), ferridoxin thioredoxin reductase (1DJ7), DNA-binding domain of HIV-1 integrase (1IHV), nitrile hydratase (1AHJ), PsaE from photosystem I protein (1PSF), ribosomal protein L14 (1WHI), C-terminal domain of ribosomal protein L2 (1RL2), Snrnp (1B34), Sso7d (1BF4), tudor domain (1G5V), myosin S1 motor domain (1D0Z) and BirA (1BIA). Under OB-fold the proteins analyzed were cold shock protein (1CSP), aspartyl t-RNA-synthetase (1ASY), heat labile enterotoxin (1LTT), mitochondrial single-stranded DNA-binding protein (3ULL), Rho protein (1A62), replication protein A (1JMC), RuvA (1CUK), ribosomal protein S12, S17 (1FJF), N-terminal domain of ribosomal protein L2 (1RL2), S1 RNA-binding domain (1SRO), staphylococcal nuclease (1EY0), T7 DNA ligase (1A0I), verotoxin-1 (1BOV), C-terminal domain of eukaryotic translation initiation factor 5a (1EIF). The protein data bank code was given in the parenthesis following the name of the protein used in the analysis. For super positioning of proteins programs from CCP4 package  were used. For graphical visualization and analysis 'O' program  was used. Comparer server  was used for structure based sequence alignment.
V.A. acknowledges a Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR), India.
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