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Bioinformatics Advance Access originally published online on September 8, 2005
Bioinformatics 2005 21(21):3959-3962; doi:10.1093/bioinformatics/bti659
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions{at}oxfordjournals.org

DNase II is a member of the phospholipase D superfamily

Iwona A. Cymerman 1, Gregor Meiss 2 and Janusz M. Bujnicki 1,*

1Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology Trojdena 4, 02-109 Warsaw, Poland
2Institute of Biochemistry, Justus-Liebig-University Giessen 35392 Giessen, Germany

*To whom correspondence should be addressed.


   Abstract
TOP
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Motivation: DNase II is an endodeoxyribonuclease involved in apoptosis and essential for the mammalian development. Despite the understanding of biochemical properties of this enzyme, its structure and relationships to other protein families remain unknown.

Results: Using protein fold-recognition we found that DNase II exhibits a catalytic domain common to the phospholipase D superfamily. Our model explains the available experimental data and provides the first structural platform for sequence–function analyses of this important nuclease.

Contact: iamb{at}genesilico.pl

Supplementary information: ftp://genesilico.pl/iamb/models/DNasell/


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Programmed cell death or apoptosis is essential for the development and homeostasis in metazoans. Defects in apoptosis cause an accumulation of abnormal cells or tissue destruction and have been associated with a number of diseases, including cancer, AIDS, ischemic strokes and neurodegeneration (reviews: Jacobson et al., 1997; Nagata, 1997). Apoptosis was originally defined by morphological criteria, including nuclear condensation of chromatin, associated with endonuclease digestion of chromosomal DNA. Several endonucleases have been implicated in this DNA cleavage, including CAD, EndoG, DNase I and DNase II (review: Counis and Torriglia, 2000).

The structural information is of key importance for apoptotic nucleases, as it allows to understand the mechanism of action at the molecular level and to interpret the functional effects of single nucleotide polymorphisms (SNPs) in the human nuclease genes that were found to correlate with defects in apoptosis. The crystal structure of DNase I has been determined (Suck et al., 1984). We modeled the 3D structure of CAD and used it as a platform to study the putative catalytic residues (Scholz et al., 2003). Our model was recently confirmed by a crystallographic analysis (Woo et al., 2004). We also predicted the structure of EndoG and validated it by mutagenesis (Schafer et al., 2004). Thus far, structural information is completely missing for DNase II (EC 3.1.22.1 [EC] ) and the molecular mechanism of its action remains largely unknown. Paradoxically, DNase II was one of the earliest endonucleases identified (Catcheside and Holmes, 1947), with considerable biochemical characterization reported already in the 1960s (Bernardi and Griffe, 1964). The activity of DNase II is distinguished from other apoptotic nucleases by its acidic pH optimum and the absence of requirement for bivalent metal ions (Evans and Aguilera, 2003). The human enzyme DNase II{alpha} is heavily glycosylated and consists of one contiguous polypeptide chain with three putative disulfide bridges. Site-directed mutagenesis implicated conserved H295 as a putative catalytic residue (MacLea et al., 2002, 2003b). Sequence analyses revealed a family of proteins conserved in metazoans as well as in a few non-metazoan species, but absent from fungi, plants or Prokaryota, with the sole exception of Burkholderia pseudomallei (MacLea et al., 2003a). However, no similarity to any other protein family could be detected, hampering sequence–structure–function studies on these enzymes.

The knowledge of sequence–structure–function relationships in DNase II is desired not only because of its involvement in apoptosis and development, but also because it is considered as a potential mucolytic agent for improving pulmonary clearance in the genetic disorder cystic fibrosis (Krieser et al., 2001). Therefore, we attempted to elucidate the structure and mechanism of action of DNase II using computational methods.


   MATERIALS AND METHODS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sequence searches of the non-redundant (nr) database were carried out at the NCBI using PSI-BLAST (Altschul et al., 1997), using the human DNase II {alpha} (GI: 3182984) as a query. Secondary structure prediction and tertiary fold recognition (FR) were carried out via the GeneSilico meta-server gateway (Kurowski and Bujnicki, 2003); references to the individual methods are provided at http://genesilico.pl/meta/). Modeling of the DNase II structure was carried out using the ‘FRankenstein's Monster’ approach for simultaneous optimization of alignments and 3D models (Kosinski et al., 2003). This method starts from crude FR alignments between the target sequence and template structures and iteratively builds homology models using MODELLER (Fiser and Sali, 2003), evaluates the local sequence–structure fit using VERIFY3D (Luthy et al., 1992) via the COLORADO3D server (Sasin and Bujnicki, 2004), realigns the best scoring fragments of models to the templates and carries out systematic realignment in the remaining poorly scored regions, restrained by the match between secondary structures predicted in the target and observed in the templates (Kosinski et al., 2003).


   RESULTS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Database searches and multiple alignment of the DNase II family revealed rather uniform sequence conservation over the entire length of the protein, with only a few regions of divergence (Fig. 1). The entire alignment as well as its two halves, corresponding to potential (sub)domains, were submitted to FR servers (see Materials and methods section for details). No FR method reported a target–template alignment with a score above the documented level of significance (http://bioinfo.pl/LiveBench/). However, for the C-terminal region, comprising residues 205–360 of the human DNase IIalpha, a number of servers, including INBGU (Fischer, 2000), FFAS03 (Rychlewski et al., 2000) and mGENTHREADER (Jones, 1999) reported the phospholipase D (PLD) fold (d.136.1.1 in SCOP) at the first position of their rankings. SPARKS (Zhou and Zhou, 2004) and 3DPSSM (Kelley et al., 2000) reported it at the second and fifth positions. All these servers identified the nuclease Nuc from Salmonella typhimurium (1byr, Stuckey and Dixon, 1999) as the preferred template, only FFAS favored PLD from Streptomyces sp. (1f0i, Leiros et al., 2004). Accordingly, the consensus selector PCONS5 (Lundstrom et al., 2001) singled out 1byr as the potentially best template for modeling the C-terminal domain (CTD) of DNase II. Examination of FR alignments revealed differences owing to sequence shifts. However, all methods found the ‘H295XK297’ motif characteristic for the active sites of PLD enzymes (Koonin, 1996; Ponting and Kerr, 1996). The multiple sequence alignment (Fig. 1) revealed another such motif (‘H113XK115’) in the N-terminal domain of DNase II (NTD).



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  		Fig. 1 Sequence alignment of representative members of the DNase II family (human enzymes {alpha} and ß and Caenorhabditis elegans Nuc-1) and their predicted relatives of known structure, Nuc and PLD. Because of space constraints, full alignment of the DNase II family is provided as a Supplementary File. Predicted catalytic residues are indicated by ‘#’, apparently missing catalytic residues are indicated by ‘?’, the alternative position of the ‘Q/D/E’ residue in the CTD is indicated by ‘*’. N and C indicate residues that are N-glycosylated or involved in disulfide bridges, respectively. Secondary structure is shown as arrows and cylinders.

 
Known members of the PLD superfamily possess a bilobed structure, with a single active site composed of two ‘H X K’ motifs located at the interface between two domains. Some of these enzymes (e.g. Nuc) are homodimers, in which each monomer contributes a half-site, but most of them (e.g. PLD) possess two mutually homologous domains within a single chain, and thereby act as pseudodimers. Thus, identification of two ‘H X K’ motifs in the DNase II sequence along with the relationship of its C-terminal domain to Nuc and PLD enzymes suggested that DNase II may be a pseudodimeric member of the PLD superfamily. However, we were unable to confirm the statistical significance of similarity between the NTD and CTD of DNase II or between the NTD and known PLD enzymes. Nonetheless, we were able to align by hand, the patterns of secondary structure predicted for both domains with each other and with the structures of Nuc and PLD (Leiros et al., 2004; Stuckey and Dixon, 1999).

In order to validate these sequence-based predictions, we constructed homology models of DNase II NTD and CTD (Fig. 2) and the corresponding dimers, based on the coordinates of Nuc (Stuckey and Dixon, 1999) and PLD (Leiros et al., 2004), and evaluated these models on the level of the 3D structure (see Materials and methods section). Isolated NTD and CTD models received acceptable VERIFY3D scores 0.30 and 0.31, slightly lower than domains of the crystal structures of Nuc (0.40) and PLD (0.43 and 0.40). The NTD–CTD and CTD–CTD dimers received scores 0.33 and 0.32, compared with 0.48 and 0.45 for the Nuc and PLD dimers, respectively. The interface residues in the NTD–CTD and CTD–CTD dimers had similar scores. Thus, according to the theoretical evaluation of the model quality, both dimers are acceptable and it is difficult to predict, which of them is more likely to be correct.



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  		Fig. 2 Model of the mature form of human DNase II{alpha}—the NTD–CTD pseudodimer variant (aa 19–361), based on the alignment shown in Figure 1. The predicted active site and disulfide bridges are indicated by sticks and balls, respectively. The image of the CTD–CTD dimer and coordinates of both models are available as Supplementary Files and from the FTP server ftp://genesilico.pl/iamb/models/DNaseII/.

 
Both models also agree equally well with the available experimental data, which were not used as modeling constraints. The known catalytic H295 residue (MacLea et al., 2003b) is located in the predicted active site in the CTD, three pairs of Cys residues found to form established disulfide bridges (C19–C159 in the NTD, and C267–C347 and C308–C327 in the CTD) are located in close proximity, whereas the known N-glycosylated residues N86, N212, N266 and N290 are expectedly located on the protein surface.


   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Known members of the PLD superfamily possess an active site composed of two HXK–Xn–N–Xn–(E/Q/D) motifs from two similar domains. One of the His residues is the first nucleophile, which attacks the phosphorus atom of the substrate and forms a covalent intermediate. In the pseudodimeric PLD enzymes, it is the His from the NTD (Leiros et al., 2004). The catalytic His from the other domain activates a water molecule being the second nucleophile. K and N residues coordinate the phosphate, whereas the D/Q/E residues coordinate the catalytic histidines.

Our finding that the CTD of DNase II is a remote relative of Nuc and PLD explains the unusual feature of this nuclease, such as the resistance to EDTA, and the ability to cleave bis(p-nitrophenyl) phosphate (Bernardi, 1971), which are also exhibited by deoxyribonucleases from the PLD superfamily, such as Nuc (Zhao et al., 1997) and the restriction enzyme BfiI (Sapranauskas et al., 2000). By similarity to PLD and Nuc, whose mechanism has been elucidated, we infer that the reaction of phosphodiester bond hydrolysis by DNase II and its relatives will proceed by a covalently linked reaction intermediate. We predict that the catalytic motif of the DNase II CTD comprises residues H295, K297 and N313. The ‘D/Q/E’ residue is replaced by G323, but we suspect that its function is taken over by an invariant D311, whose carboxyl group can assume an equivalent position in space. There is no statistical support from database searches for the homology of the NTD to PLD, but our hand-guided alignment suggests a second catalytic motif that may comprise residues H113, K115 and Q155. However, we were unable to identify a conserved counterpart of the Asn residue in the NTD either in the sequence or at the structural level.

The hypothesis that the NTD may be also a PLD superfamily member suggests that H113 rather than H295 could be the first nucleophile that forms the phosphohistidine moiety. Based on theoretical considerations, presently we cannot confidently discriminate between the alternative models of the active site of DNase II being formed by the CTD–CTD dimer or the NTD–CTD pseudodimer. However, the models presented in this work offer a convenient platform not only for testing the hypothesis concerning the oligomeric state of the enzyme, but also suggest a number of residues to be studied by site-directed mutagenesis and allow to ask precise questions about their possible roles in the reaction mechanism.


   Acknowledgments
 
This analysis was funded by the NIH (grant R03 TW007163-01). J.M.B. is an EMBO/HHMI Young Investigator. I.A.C. is a recipient of a scholarship from the Postgraduate School of Molecular Medicine at the Medical University of Warsaw.

Conflicts of Interest: none declared.

Received on February 25, 2005; revised on July 16, 2005; accepted on September 1, 2005

   REFERENCES
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

    Altschul, S.F., et al. (1997) Gapped BLAST and PSI-BLAST. Nucleic Acids Res., 25, 3389–3402[Abstract/Free Full Text].

    Bernardi, G. (1971) Spleen acid deoxyribonuclease. In Boyer, P.D. (Ed.). The Enzymes, , NY Academic Press, pp. 271–287.

    Bernardi, G. and Griffe, M. (1964) Studies on acid deoxyribonuclease. II. Isolation and characterization of spleen-acid deoxyribonuclease. Biochemistry, 20, 1419–1426.

    Catcheside, D.G. and Holmes, B. (1947) The action of enzymes on chromosomes. Symp. Soc. Exp. Biol., 1, 225–231.

    Counis, M.F. and Torriglia, A. (2000) DNases and apoptosis. Biochem. Cell Biol., 78, 405–414[CrossRef][Web of Science][Medline].

    Evans, C.J. and Aguilera, R.J. (2003) DNase II: genes, enzymes and function. Gene, 322, 1–15[CrossRef][Web of Science][Medline].

    Fischer, D. (2000) Hybrid fold recognition: combining sequence derived properties with evolutionary information. Pacific Symp. Biocomp., 119–130.

    Fiser, A. and Sali, A. (2003) Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol, 374, 461–491[Web of Science][Medline].

    Jacobson, M.D., et al. (1997) Programmed cell death in animal development. Cell, 88, 347–354[CrossRef][Web of Science][Medline].

    Jones, D.T. (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J. Mol. Biol., 287, 797–815[CrossRef][Web of Science][Medline].

    Kelley, L.A., et al. (2000) Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol., 299, 499–520[Web of Science][Medline].

    Koonin, E.V. (1996) A duplicated catalytic motif in a new superfamily of phosphohydrolases and phospholipid synthases that includes poxvirus envelope proteins. Trends Biochem. Sci., 21, 242–243[CrossRef][Web of Science][Medline].

    Kosinski, J., et al. (2003) A ‘FRankenstein's monster’ approach to comparative modeling. Proteins, 53, Suppl. 6, 369–379[CrossRef][Web of Science][Medline].

    Krieser, R.J., et al. (2001) The cloning, genomic structure, localization, and expression of human deoxyribonuclease IIbeta. Gene, 269, 205–216[CrossRef][Web of Science][Medline].

    Kurowski, M.A. and Bujnicki, J.M. (2003) GeneSilico protein structure prediction meta-server. Nucleic Acids Res., 31, 3305–3307[Abstract/Free Full Text].

    Leiros, I., et al. (2004) The reaction mechanism of phospholipase D from Streptomyces sp. strain PMF. J. Mol. Biol., 339, 805–820[CrossRef][Web of Science][Medline].

    Lundstrom, J., et al. (2001) Pcons: a neural-network-based consensus predictor that improves fold recognition. Protein Sci., 10, 2354–2362[CrossRef][Web of Science][Medline].

    Luthy, R., et al. (1992) Assessment of protein models with three-dimensional profiles. Nature, 356, 83–85[CrossRef][Medline].

    MacLea, K.S., et al. (2002) Revised structure of the active form of human deoxyribonuclease II{alpha}. Biochem. Biophys. Res. Commun., 292, 415–421[CrossRef][Web of Science][Medline].

    MacLea, K.S., et al. (2003a) A family history of deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei. Gene, 305, 1–12[CrossRef][Web of Science][Medline].

    MacLea, K.S., et al. (2003b) Structural requirements of human DNase II alpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging. Biochem. J., 371, 867–876[CrossRef][Web of Science][Medline].

    Nagata, S. (1997) Apoptosis by death factor. Cell, 88, 355–365[CrossRef][Web of Science][Medline].

    Ponting, C.P. and Kerr, I.D. (1996) A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases. Protein Sci., 5, 914–922[Web of Science][Medline].

    Rychlewski, L., et al. (2000) Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Sci., 9, 232–241[Web of Science][Medline].

    Sapranauskas, R., et al. (2000) Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium. J. Biol. Chem., 275, 30878–30885[Abstract/Free Full Text].

    Sasin, J.M. and Bujnicki, J.M. (2004) COLORADO3D, a web server for the visual analysis of protein structures. Nucleic Acids Res, 32, W586–589[Abstract/Free Full Text].

    Schafer, P., et al. (2004) Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis. J. Mol. Biol., 338, 217–228[CrossRef][Web of Science][Medline].

    Scholz, S.R., et al. (2003) Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase. Biochemistry, 42, 9288–9294[CrossRef][Medline].

    Stuckey, J.A. and Dixon, J.E. (1999) Crystal structure of a phospholipase D family member. Nat. Struct. Biol., 6, 278–284[CrossRef][Web of Science][Medline].

    Suck, D., et al. (1984) Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution. EMBO J, 3, 2423–2430[Web of Science][Medline].

    Woo, E.J., et al. (2004) Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway. Mol. Cell, 14, 531–539[CrossRef][Web of Science][Medline].

    Zhao, Y., et al. (1997) Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases. Protein Sci., 6, 2655–2658[Web of Science][Medline].

    Zhou, H. and Zhou, Y. (2004) Single-body residue-level knowledge-based energy score combined with sequence-profile and secondary structure information for fold recognition. Proteins, 55, 1005–1013[CrossRef][Web of Science][Medline].


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This Article
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