A homology model of restriction endonuclease SfiI in complex with DNA
© Chmiel et al; licensee BioMed Central Ltd. 2005
Received: 08 October 2004
Accepted: 24 January 2005
Published: 24 January 2005
Restriction enzymes (REases) are commercial reagents commonly used in recombinant DNA technologies. They are attractive models for studying protein-DNA interactions and valuable targets for protein engineering. They are, however, extremely divergent: the amino acid sequence of a typical REase usually shows no detectable similarities to any other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. From structural analyses and bioinformatics studies it has been learned that some REases belong to at least four unrelated and structurally distinct superfamilies of nucleases, PD-DxK, PLD, HNH, and GIY-YIG. Hence, they are extremely hard targets for structure prediction and homology-based inference of sequence-function relationships and the great majority of REases remain structurally and evolutionarily unclassified.
SfiI is a REase which recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3' and generates 3 nt long 3' overhangs upon cleavage. SfiI is an archetypal Type IIF enzyme, which functions as a tetramer and cleaves two copies of the recognition site in a concerted manner. Its sequence shows no similarity to other proteins and nothing is known about the localization of its active site or residues important for oligomerization. Using the threading approach for protein fold-recognition, we identified a remote relationship between SfiI and BglI, a dimeric Type IIP restriction enzyme from the PD-DxK superfamily of nucleases, which recognizes the 5'GCCNNNN^NGGC3' sequence and whose structure in complex with the substrate DNA is available. We constructed a homology model of SfiI in complex with its target sequence and used it to predict residues important for dimerization, tetramerization, DNA binding and catalysis.
The bioinformatics analysis suggest that SfiI, a Type IIF enzyme, is more closely related to BglI, an "orthodox" Type IIP restriction enzyme, than to any other REase, including other Type IIF REases with known structures, such as NgoMIV. NgoMIV and BglI belong to two different, very remotely related branches of the PD-DxK superfamily: the α-class (EcoRI-like), and the β-class (EcoRV-like), respectively. Thus, our analysis provides evidence that the ability to tetramerize and cut the two DNA sequences in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily of REases. The model of SfiI will also serve as a convenient platform for further experimental analyses.
Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: ). The "orthodox" Type IIP REases are dimeric, they recognize a short (4–8 bp) palindromic sequence of double-stranded DNA and in the presence of Mg2+, catalyze the hydrolysis of phosphodiester bonds at precise positions within or close to this sequence, leaving "blunt" or "sticky" ends (with a 5' or 3' overhangs). The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: ). REases coupled with DNA methyltransferases (MTases) of similar specificity form restriction-modification (RM) systems, which are ubiquitous among Bacteria and Archaea . While cleavage at specific sequences provides efficient means of destroying foreign DNA, methylation of these sequences in the prokaryotic chromosome renders them resistant to REase and thereby protects the own DNA from cleavage. Because cleavage of the chromosomal DNA in unmodified sequences would be deletorious for the cell, the REases must maintain extremely high specificities, tightly coupled with that of the methyltransferase. A change in just one base pair of the "cognate" site can reduce the ratio kcat/Km for DNA cleavage by a factor ≥106 .
To date, only crystal structures of 15 REases have been solved, compared to over 3000 biochemically characterized enzymes (review: ; see also  for updates). It was found that they share a characteristic structural core and a very weakly conserved catalytic motif (P)D-Xn-(D/E)-X-K (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity (review: ). The comparison of crystal structures of members of this so-called "PD-DxK" superfamily suggested that the catalytic and DNA-binding regions are major determinants of structural stability of these proteins . Structural comparisons revealed also two major branches or classes, α and β, whose archetypal members were the enzymes that cleave DNA to generate 4 nt long 5' "sticky ends", for instance EcoRI (α-class), and those that generate "blunt" ends after the cleavage, for instance EcoRV (β-class) [7–9]. Using the experimentally solved structures as templates, bioinformatics methods such as iterative sequence database searches and protein fold-recognition have been used to predict the active site in some REases [10–13]. From these analyses it was learned that certain functional peculiarities, like the requirement of a binding of an uncleaved effector site for cleavage of another site characteristic for Type IIE enzymes, evolved independently in the α and β branches of the PD-DxK superfamily [11, 13–15].
It was also found that other REases belong to completely unrelated superfamilies, with different three-dimensional folds and catalytic sites (review: ): BfiI is a member of the phospholipase D (PLD) superfamily , Eco29kI belongs to the GIY-YIG superfamily , and KpnI and a few other REases belong to the HNH superfamily [17–19]. While the structural information is essential to infer the molecular basis of sequence specificity in REases, the lack of overall sequence conservation in these enzymes, the absence of invariable residues even in the active site and the presence of several alternative folds make structure prediction and classification extremely difficult.
SfiI is a REase isolated from Streptomyces fimbriatus. It recognizes the interrupted palindromic sequence 5'GGCCNNNN^NGGCC3', where N denotes any base, and cleaves it as indicated by "^", leaving 3' extensions 3 nt long . SfiI is a prototype of Type IIF enzymes, which function as tetramers that bind simultaneously to two recognition sites and cleave both sites concertedly . However, a structural model of SfiI, which could be used as a platform to study its sequence-function relationships, is not yet available. Thus, despite the availability of a large body of biochemical data on how SfiI interacts with the DNA substrate (mainly on the kinetics of protein-DNA interactions with different substrates and the geometry of DNA looping [22–25], but not on the "residue-level" details thereof), the identity of amino acid residues important for dimerization, tetramerization, DNA binding and cleavage remains completely unknown. We have therefore carried out bioinformatics analyses of SfiI that allowed to identify its closest relative amongst REases with known structure and use this information to construct a tertiary model of SfiI in complex with its target DNA.
Results and discussion
In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes . Homology-modeled structures may be of too low resolution to characterize the protein-protein or protein-DNA contacts at the atomic level, but they can suggest which sequence regions or individual amino-acids are essential components of the binding surfaces. In particular, identification of amino acids potentially involved in protein-DNA contacts may guide mutagenesis experiments aimed at the engineering protein variants with novel specificities. However, homology modeling requires a homologous template structure to be identified and the sequence of the protein of interest (a target) to be correctly aligned to the template.
Identification of the three-dimensional fold of SfiI
The sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations (match of secondary structure elements, compatibility of residue-residue contacts, etc.) (reviews: [27, 28]). The SfiI sequence was therefore submitted to the GeneSilico protein fold-recognition metaserver . As expected, fold-recognition methods that rely only on sequence similarity (PDB-BLAST, and FFAS) failed to identify any significant matches between SfiI and proteins with known structrues. However, several threading methods that explicitly use the structural information from the templates reported a match between SfiI and the structure of a Type II REase BglI , a member of the PD-DxK superfamily of nucleases (FUGUE : 4.25, INBGU : 3.8, SAM-T02 : 0.13, 3DPSSM : 4.4; note that these scores are not normalized as each server uses a different evaluation system; see the individual references for details). Additionally, FUGUE reported a match (low score 3.17) between SfiI and the structure of another REase, EcoRV . Despite the scores reported by the individual threading methods (except FUGUE for BglI) were hardly significant, the consensus server Pcons5  assigned a significant score (1.35) to the BglI structure as a potential modeling template.
Homology modeling of the SfiI monomer
Modeling of the SfiI dimer in complex with the DNA
Model-based identification of amino acid residues important for catalysis, DNA-binding and dimerization of SfiI
Interestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63–80 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities.
The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge (E63R, E66R, R73D, F74R) could disrupt the formation of the SfiI dimer and destroy the REase activity.
Prediction of the dimer-dimer interaction surface in the SfiI tetramer
Implications for the evolutionary history of different (sub) Types of REases
Comparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: α (EcoRI-like) and β (EcoRV-like) [7–9]. It was proposed that extant REases evolved independently from non-specific or structure-specific nucleases from both lineages (review: ). Interestingly, the phylogenetic tree of the PD-DxK superfamily revealed intriguing cases of convergent evolution. So far, it was found that Type IIE enzymes that bind two copies of the recognition site (the actual target of cleavage and the non-cleaved allosteric effector), evolved independently at least three times: EcoRII is an α-lineage member that apparently evolved from IIP enzymes similar to SsoII or PspGI by acquisition of an N-terminal effector-binding domain [12, 42]. NaeI is a β-lineage member remotely related to IIP enzymes EcoRV and HincII that acquired a C-terminal effector-binding domain unrelated to that of EcoRII . Finally, Sau3AI is a β-lineage member that apparently evolved by a duplication of a catalytic domain closely related to a DNA repair enzyme MutH, followed by the loss of catalytic residues in the C-terminal domain, thereby adapted to function as an effector-binding domain [14, 15]. Another type of evidence for convergent evolution is provided by the finding that the specificity for the GATC sequence appeared independently in the α-lineage (MboI and its close homologs [5, 13]) and in the β-lineage (Sau3AI and its close homologs [14, 15]).
Our results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a β-lineage member, an "orthodox" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises α-lineage members for which crystal structures were solved (NgoMIV, Cfr10I, Bse643I) [39–41]. Thus, our analysis provides evidence that the ability of Type IIF REases to tetramerize and cut two target sites in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily. Different Type IIF REases appear to have evolved independently from the simplest, "orthodox" Type IIP enzymes, like previously found for Type IIE REases. It was previously demonstrated that deletion of the effector-binding domain converts the Type IIE REase EcoRII to function as a Type IIP enzyme . However, tetramerization seems to be important for the catalytic activity of the Type IIF enzyme Cfr10I, since the DNA cleavage activity of the dimeric W220A mutant of this REase is <0.1% of that of the wild-type enzyme . In the absence of a high resolution co-crystal structure of SfiI in complex with DNA, our model will serve as a convenient platform to study sequence-structure-function relationships in this enzyme. In particular, it will facilitate the mutagenesis of residues potentially involved in tetramerization, dimerization, DNA-binding and catalysis.
Sequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST . Secondary structure prediction and tertiary fold-recognition was carried out via the GeneSilico meta-server gateway . Secondary structure prediction was predicted using PSIPRED , PROFsec , PROF , SABLE , JNET , JUFO , and SAM-T02 . Solvent accessibility for the individual residues was predicted with SABLE  and JPRED . The fold-recognition analysis (attempt to match the query sequence to known protein structures) was carried out using FFAS03 , SAM-T02 , 3DPSSM , INBGU , FUGUE , mGENTHREADER , and SPARKS . Fold-recognition alignments reported by these methods were compared, evaluated, and ranked by the Pcons server .
The alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the "FRankenstein's Monster" approach , comprising cycles of model building by MODELLER , evaluation by VERIFY3D  via the COLORADO3D server , realignment in poorly scored regions and merging of best scoring fragments. The positions of predicted catalytic residues and secondary structure elements were used as spatial restraints. This strategy has previously helped us to build accurate, experimentally validated models of other REases, such as SsoII , PspGI , MboI , and KpnI .
List of abbreviations
product of an open reading frame
This analysis was funded by KBN (grant 3P04A01124 to JMB). The work of KJS and AAC was supported by the NIH (Fogarty International Center grant R03 TW007163-01). JMB was supported by the EMBO/HHMI Young Investigator Award and by the Fellowship for Young Scientists from the Foundation for Polish Science. We would like to thank Alfred Pingoud and Virgis Siksnys for stimulating discussions concerning the evolution of sequence-structure-function relationships in REases of various subTypes.
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