Dual Role for Zn2+ in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease
2007; Elsevier BV; Volume: 282; Issue: 44 Linguagem: Inglês
10.1074/jbc.m705927200
ISSN1083-351X
AutoresSaravanan Matheshwaran, Kommireddy Vasu, Soumitra Ghosh, Valakunja Nagaraja,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoWe describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase. We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase. A large number of proteins have bound zinc ions, which contribute to protein stability and/or catalytic functions more widely than any other transition metal ions (1Coleman J.E. Annu. Rev. Biochem. 1992; 61: 897-946Crossref PubMed Scopus (858) Google Scholar, 2Vallee B.L. Kim D.S. Biochemistry. 1990; 29: 5647-5659Crossref PubMed Scopus (1536) Google Scholar). A catalytic role for zinc was first shown in the case of carbonic anhydrase (3Keilin D. Kim T. Biochem. J. 1940; 34: 1163-1176Crossref PubMed Google Scholar), and its structural role was first proposed and demonstrated for the transcription factor TFIIIA (4Hanas J.S. Kim D.J. Bogenhagen D.F. Wu F.Y. Wu C.W. J. Biol. Chem. 1983; 258: 14120-14125Abstract Full Text PDF PubMed Google Scholar, 5Miller J. Kim A.D. Klug A. EMBO J. 1985; 4: 1609-1614Crossref PubMed Scopus (1705) Google Scholar). Since then, the roles for Zn2+ in numerous zinc-binding proteins have been identified and characterized. In many examples, the role of zinc ion is neither strictly structural nor catalytic, as in aminoacyl-tRNA synthetases, where zinc is involved in amino acid discrimination (6Sankaranarayanan R. Kim A.C. Rees B. Bovee M. Caillet J. Romby P. Francklyn C.S. Moras D. Nat. Struct. Biol. 2000; 7: 461-465Crossref PubMed Scopus (142) Google Scholar). Zinc binding motifs are structurally diverse and are present among proteins that perform a broad range of functions in various cellular processes. For instance, the motifs play a role in DNA recognition, transcription activation, protein folding and assembly, and protein-protein interactions (7Laity J.H. Kim B.M. Wright P.E. Curr. Opin. Struct. Biol. 2001; 11: 39-46Crossref PubMed Scopus (1096) Google Scholar). Zinc binding is observed in different groups of nucleases, I-PpoI, I-TevI, T4 endonuclease VII, DNA repair endonuclease IV, colicin E7, and S1 nuclease (8Flick K.E. Kim M.S. Monnat R.J. Jr. Stoddard B. L. Nature. 1998; 394: 96-101Crossref PubMed Scopus (195) Google Scholar, 9Van Roey P. Kim C.A. Fox K.M. Belfort M. Derbyshire V. EMBO J. 2001; 20: 3631-3637Crossref PubMed Scopus (62) Google Scholar, 10Hosfield D.J. Kim Y. Haas B.J. Cunningham R.P. Tainer J.A. Cell. 1999; 98: 397-408Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 11Ko T.P. Kim C.C. Ku W.Y. Chak K.F. Yuan H.S. Structure. 1999; 7: 91-102Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 12Gite S. Kim V. Eur. J. Biochem. 1992; 210: 437-441Crossref PubMed Scopus (18) Google Scholar). The binding of zinc is important for structural stability of I-PpoI, I-TevI, and T4 endonuclease VII and for catalysis in endonuclease IV and colicin E7. Bioinformatic analysis showed that McrA has a zinc binding fold, suggested to be needed for structural integrity (13Bujnicki J.M. Kim M. Rychlewski L. Mol. Microbiol. 2000; 37: 1280-1281Crossref PubMed Scopus (22) Google Scholar). R.BslI contains two glucocorticoid receptor-like zinc (Cys4) binding motifs, which are important for the protein-DNA and protein-protein interactions (14Vanamee E.S. Kim P. Zhu Z. Yates D. Garman E. Xu S. Aggarwal A.K. J. Mol. Biol. 2003; 334: 595-603Crossref PubMed Scopus (8) Google Scholar). In this paper, we describe two distinct roles for Zn2+ in R.KpnI. Type II REases 3The abbreviations used are: REase, restriction endonuclease; WT, wild type. require Mg2+ or a similar divalent metal ion to cleave DNA. Almost 3700 Type II restriction enzymes, representing more than 262 distinct specificities, are known to date (15Roberts R.J. Kim T. Posfai J. Macelis D. Nucleic Acids Res. 2007; 35: D269-D270Crossref PubMed Scopus (207) Google Scholar). Most Type II REases belong to the PD... (D/E)XK superfamily (16Pingoud A. Kim M. Pingoud V. Wende W. Cell Mol. Life Sci. 2005; 62: 685-707Crossref PubMed Scopus (376) Google Scholar). Recent structural and bioinformatics studies revealed that apart from the PD... (D/E)XK superfamily, few REases belong to other nuclease superfamilies, such as Nuc, HNH, and YIG-GIY, which are structurally unrelated to each other (17Sapranauskas R. Kim G. Lagunavicius A. Vilkaitis G. Lubys A. Siksnys V. J. Biol. Chem. 2000; 275: 30878-30885Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Aravind L. Kim K.S. Koonin E.V. Nucleic Acids Res. 2000; 28: 3417-3432Crossref PubMed Google Scholar, 19Bujnicki J.M. Kim M. Rychlewski L. Trends Biochem. Sci. 2001; 26: 9-11Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Sequence alignment and subsequent validation experiments showed that R.KpnI is the first member of the HNH superfamily (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar). Although at first glance R.KpnI appeared to be a typical dimeric Type IIP REase recognizing and cleaving palindromic sequence GGTACC, it has several distinct features. The properties include prolific promiscuous activity in the presence of Mg2+ which is further enhanced with Mn2+, efficient site specific high fidelity DNA cleavage when Ca2+ is used instead of Mg2+, and suppression of the promiscuous cleavage activity in presence of Ca2+ (21Chandrashekaran S. Kim M. Radha D.R. Nagaraja V. J. Biol. Chem. 2004; 279: 49736-49740Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Kinetic studies revealed that the Ca2+-mediated exquisite specificity is achieved at the step of DNA cleavage (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). The alignment of McrA, T4 endonuclease VII, and R.KpnI is depicted in Fig. 1A. The former two enzymes have tetra-Cys Zn2+ fingers, whereas R.KpnI has an unusual CCCH putative Zn2+ finger. Here we describe the importance of the Zn2+ finger motif in Zn2+ coordination. Surprisingly, the bound Zn2+ has more complex, multiple roles in R.KpnI function, in a manner distinct from any other restriction-modification system. Enzymes and DNA—T4 polynucleotide kinase, Pfu DNA polymerase, and DpnI were purchased from New England Biolabs. Oligonucleotides (Sigma and Microsynth) were purified on 18% urea-polyacrylamide gel (33Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and end-labeled with T4 polynucleotide kinase and [γ-32P]ATP (6000 Ci/mmol). Mutagenesis, Expression, and Purification of Mutant Proteins—The model of R.KpnI was built using the structure of T4 endonuclease VII and other structurally characterized HNH superfamily nucleases. The detailed procedure has been described and discussed previously (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar). Site-directed mutagenesis was performed by the megaprimer method (23Kirsch R.D. Kim E. Nucleic Acids Res. 1998; 26: 1848-1850Crossref PubMed Scopus (206) Google Scholar). The mutations were confirmed by sequencing. The WT and mutants were expressed in Escherichia coli BL26 (F-omp T hsdSB (rB- mB-) gal dcm Δlac (DE3) nin5 lac UV5-T7 gene 1) containing KpnI methyltransferase, and the cells were induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside as described previously (24Chandrashekaran S. Kim P. Nagaraja V. J. Biosci. 1999; 24: 269-277Crossref Scopus (14) Google Scholar). Cells were lysed by sonication in buffer A containing 10 mm potassium phosphate (pH 7.0), 1 mm EDTA, 7 mm 2-mercaptoethanol. The supernatant was subjected to 0-50% ammonium sulfate fractionation. The samples were dialyzed against buffer A and purified by phosphocellulose and Hi-Trap heparin columns. The fractions containing the enzyme were pooled and dialyzed against buffer B (10 mm Tris-HCl (pH 7.4), 0.1 mm EDTA, 50 mm KCl, 5 mm 2-mercaptoethanol, and 50% glycerol). The concentration of the proteins was estimated by the method of Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Atomic Absorption Analysis—Purified R.KpnI (10 mg) was denatured and renatured in the presence or absence of 100 μm ZnCl2 or 5 mm EDTA and then dialyzed overnight against 20 mm Tris-HCl (pH 7.4), 150 mm NaCl at 4 °C with buffer changes to eliminate excess metal ions or chelators. Chelex-100 resin (Sigma) was used to remove trace metal ions in all of the buffers. The samples were analyzed by atomic absorption spectroscopy. The dialyzed buffer after Chelex treatment was used as a blank, and the residual Zn2+ background was subtracted from the measurement of protein samples. Zn2+ Blotting Assay—Purified R.KpnI and its mutants (0-6 μg) was slot-blotted onto nitrocellulose membrane presoaked in buffer (10 mm Tris, pH 7.5, 100 mm NaCl, 1 mm EDTA). Proper transfer was ascertained by Ponceau-S staining with transferred protein amounts estimated using Quantity One software. After transfer, the membrane was incubated at 37 °C for 1 h in buffer containing 10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm MgCl2 and subsequently washed three times (15 min each) in the same buffer. The membrane was next incubated in buffer containing 30 μCi of 65ZnCl2 (specific activity, 800 mCi/g; BARC, Mumbai) at room temperature for 1 h with gentle rocking. The unbound radioactivity was removed by washing the membrane three times with buffer (20 min each). The membrane was dried and exposed to a PhosphorImager screen. Electrophoretic Mobility Shift Assay—Different concentrations of the WT (1-10 nm) and mutant R.KpnI (10-100 nm) were incubated with the 0.2 pmol of end-labeled double-stranded oligonucleotide (20-mer) containing the R.KpnI recognition site in binding buffer (20 mm Tris-HCl (pH 7.4), 25 mm NaCl, and 5 mm 2-mercaptoethanol) for 15 min on ice. The free DNA and the enzyme-bound complexes were resolved by 8% native polyacrylamide gel electrophoresis in 1× TBE buffer (89 mm Tris-HCl, 89 mm boric acid, and 1 mm EDTA), and then signals were detected by autoradiography. In Vitro DNA Cleavage and Steady-state Kinetic Analysis—Purified R.KpnI and its mutants were incubated with 500 ng of plasmid DNA in buffer containing 10 mm Tris-HCl (pH 7.4), 5 mm 2-mercaptoethanol, 2 mm MgCl2, or 10-100 μm ZnCl2 for 1 h at 37 °C. The cleavage products were analyzed on 1% agarose gel. For kinetic analysis, the purified enzyme was dialyzed against 10 mm EDTA to remove any bound metal ions. Steady-state kinetic time courses with canonical DNA substrates were measured at DNA concentrations of 5-150-fold molar excess over dimeric enzyme (1 nm) in the presence of 100 μm Zn2+. The kinetic parameters were determined as described (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). Circular Dichroism—The wild type R.KpnI and its mutants harboring the C119A, C128A, C171A, and H174A mutations were analyzed by CD. The CD spectra were recorded at 25 °C from 250 to 200 nm using a JASCO J-720 spectropolarimeter and a cuvette of path length 0.2 cm. The spectra were collected at scanning rate of 50 nm/min, and triplicate spectrum readings were collected per sample. All of the samples were base line-corrected before calculations. The buffer used was 50 mm Tris-HCl (pH 7.4), 75 mm NaCl, 1 mm 2-mercaptoethanol. The proteins were at a concentration of 0.2 μg/μl, and the molar ellipticity (θ) was calculated using the equation, θ=θobs×10−3×MrC×l×n×10−2deg dmol−1 cm2 where θobs is the observed ellipticity, Mr is molecular weight, C is concentration (in mg/ml), l is the path length of the cuvette in centimeters, n refers to the number of residues, and deg is degrees. Thermal stability of the protein samples was assessed using CD by following changes in the spectrum with increasing temperature (25-75 °C). A single wavelength (222 nm) was chosen to monitor the protein structure, and the signal at that wavelength is recorded continuously as the temperature is raised. Tryptic Digestion of R.KpnI—Proteolytic digestions of different samples of R.KpnI and its mutants (1 mg/ml) were carried out in 50 mm Tris-HCl buffer, pH 8.5, and at 37 °C using 1% trypsin. 5-μl samples (5 μg) were taken after various time periods, and trypsin was inactivated with buffer containing phenylmethylsulfonyl fluoride. Samples were analyzed by 15% SDS-PAGE. R.KpnI Has Two Zn2+ Binding Sites—Sequence analysis and homology modeling of R.KpnI predicted the presence of an unusual CCCH zinc finger motif (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar) different from other previously described commonly found zinc finger motifs (CCCC, CCHH, CCHC). The putative zinc-coordinating residues are shown in the model (Fig. 1B). The arrangement of cysteines and histidine (CCCH) in R.KpnI is rare among zinc finger proteins of prokaryotic origin. To estimate the bound Zn2+, weper-formed atomic absorption spectrometry (Table 1). Extensively dialyzed R.KpnI was found to bind 2 mol of Zn2+/mol of dimer. The dimeric nature of the enzyme has been established before (24Chandrashekaran S. Kim P. Nagaraja V. J. Biosci. 1999; 24: 269-277Crossref Scopus (14) Google Scholar). Bound Zn2+ was not replaceable with other metal ions, since even after exhaustive dialysis against a buffer that contained 10 mm MgCl, 2 mol of Zn2+ were still retained in the protein, indicating that the site was inert to exchange by Mg2+. However, when urea-denatured protein was renatured in presence of ZnCl2, the zinc content increased to 4 mol/mol of R.KpnI dimer. Dialysis of this preparation in the presence of MgCl2 resulted in loss of 2 mol of Zn2+ from R.KpnI, indicating the replacement of two of the four Zn2+ ions by Mg2+. The other 2 mol of tightly bound zinc could not be replaced by Mg2+. These results show that R.KpnI monomer possesses two zinc-binding sites; one is replaceable with Mg2+, and another one is not (Table 1).TABLE 1Zn2+ atomic absorption spectroscopy of R.KpnIR.KpnISample preparationZn2+[Zn2+]/[E]μmμm5.0Chelex-treated10.32 ± 0.42.16 ± 0.085.0100 μm ZnCl219.54 ± 1.23.96 ± 0.065.02 mm MgCl29.45 ± 0.41.92 ± 0.085.010 mm EDTA9.74 ± 0.41.95 ± 0.05.08 m urea0.20 ± 0.20.04 ± 0.0 Open table in a new tab Role of the Zn2+ in Structural Integrity—To define the role of zinc atoms in R.KpnI, we compared the DNA binding and cleavage properties of native, zinc-demetalated (renatured in the absence of Zn2+), and zinc-reconstituted enzymes. The zinc-demetalated R.KpnI (the apoenzyme with no Zn2+ bound) did not bind DNA (Fig. 2A). The enzyme had no DNA cleavage activity in the presence of 2 mm MgCl2. Similar experiments were carried out with native and zinc reconstituted R.KpnI (Fig. 2B). The zinc-reconstituted enzyme binds and cleaves the DNA in a promiscuous manner in Mg2+-catalyzed reactions, similar to the native enzyme. To investigate whether the loss of DNA binding and cleavage in Zn2+-demetalated R.KpnI is due to the structural alterations in the protein, we carried out CD analysis. In presence of Zn2+, the far-UV CD spectrum of R.KpnI has two negative maxima at 208 and 222 nm, which is a characteristic of helical conformation. The zinc-demetalated enzyme showed altered secondary structure compared with zinc-reconstituted enzyme, indicating the importance of Zn2+ coordination to maintain the secondary structure of the R.KpnI (Fig. 3A). The stability of these proteins was monitored by CD thermal denaturation. Unfolding profiles were measured at 222 nm, from 30 to 75 °C. The Tm of zinc-reconstituted enzyme was increased by ∼8 °C over the zinc-demetalated enzyme, indicating a role for Zn2+ in stability of the enzyme (Fig. 3B). In accordance with CD spectroscopy and thermal melting experiments, proteolytic experiments also showed that the zinc-demetalated enzyme is more susceptible to trypsin cleavage than the native or zinc-reconstituted enzyme (Fig. 3C). We conclude that Zn2+ is required for stabilization of the enzymatically active R.KpnI conformation.FIGURE 3Structural alterations in R.KpnI. A, CD spectra of R.KpnI. Shown are the mean residue ellipticity of CD spectra for demetalated (dotted line) and zinc-reconstituted R.KpnI (continuous line). Changes in secondary structure were monitored by scanning from 200 to 250 nm. B, temperature-dependent CD measurements (222 nm) for demetalated (dotted line) and zinc-reconstituted R.KpnI (continuous line). All spectra represent the average of three scans using protein concentrations of 0.25 mg/ml. C, trypsin digestion profiles of zinc-reconstituted and zinc-demetalated R.KpnI. The detailed procedure is described under "Experimental Procedures." The digestion profiles were resolved on 12% SDS-PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The CCCH Motif Is Involved in Zn2+ Coordination and Maintenance of the R.KpnI Structure—To establish that the zinc binding is through the unusual zinc finger motif shown in Fig. 1A, point mutations were generated in R.KpnI. The cysteines (Cys119, Cys128, and Cys171) and histidine (His174) of the putative motif were individually changed into alanine by site-directed mutagenesis. The mutant proteins were analyzed for radioactive zinc binding using a zinc blotting assay. All of the alanine replacement mutants failed to bind radioactive zinc (Fig. 4A) in contrast to R.KpnI. Zn2+ has been shown to be essential for the folding and stability of many Zn2+ finger proteins. Zinc blotting experiments indicate that Cys119, Cys128, Cys171, and His174 are responsible for coordinating Zn2+ in R.KpnI. We examined the effect of impairment in Zn2+ coordination on the stability of the enzyme by monitoring the CD thermal melting curves of the alanine replacement mutant proteins. The normalized CD absorbance at 222 nm as a function of temperature for WT and mutants is shown in Fig. 4B. The mutant proteins showed decreased thermal stability compared with that of the WT R.KpnI, indicating that the mutations at the CCCH motif affect the folding of the enzyme. Further, the mutant enzymes showed increased protease susceptibility compared with R.KpnI, confirming the importance of the CCCH motif for the structural stability of the enzyme (Fig. 4C). Effect of CCCH Zn2+ Finger Mutations on DNA Cleavage and Binding—R.KpnI and its mutants were analyzed for the ability to cleave DNA. Under the assay conditions, wherein pUC18 DNA was completely cleaved by 1 nm WT enzyme, there was no cleavage product observed with all the four mutants even at a 100-fold excess of the enzyme (Fig. 4D). To address the cause for the loss of DNA cleavage observed with the mutants, we analyzed the DNA binding ability of the mutants by electrophoretic mobility shift assay. The mutants failed to bind the DNA containing R.KpnI recognition sequence (Fig. 4E). The mutants showed no detectable DNA binding and cleavage, due to the loss of the structure as observed in CD thermal melting and proteolytic experiments. These results suggest that the loss of coordination with zinc affected the structural integrity of the protein, concomitantly affecting the activity of the enzyme. The loss of DNA binding and cleavage seen with single amino acid substitution in R.KpnI is a typical characteristic of Zn2+ finger proteins where Zn2+ has a structural role. Specific DNA Cleavage and Suppression of Mg2+- and Mn2+-induced Promiscuous Activity—The atomic absorption spectroscopy analysis of zinc-reconstituted R.KpnI showed that the enzyme binds 4 mol of zinc (Table 1). Among the 4 mol, only 2 mol can be readily replaced with Mg2+. This hints at the possibility of zinc ions binding to the active site to influence the enzymatic properties of R.KpnI in addition to the tight coordination at the CCCH motif. The additional 2 mol of Zn2+ bound replacing the Mg2+ may inhibit DNA cleavage. Surprisingly, the enzyme showed efficient DNA cleavage in the presence of 50 μm Zn2+ (Fig. 5A). To evaluate the role of Zn2+ in the specificity of R.KpnI, we carried out DNA cleavage experiments at higher enzyme concentrations (50-1000 nm) and in the presence of 100 μm Zn2+. Even at such high concentrations of the enzyme, promiscuous cleavage is not detected, unlike in Mg2+-catalyzed reactions (Fig. 5B). In experiments using one of the noncanonical oligonucleotides (GtTACC) as a substrate in the presence of 2 mm Mg2+ or 100 μm Zn2+, the cleavage was observed only with Mg2+, indicating that the enzyme is highly specific in the presence of Zn2+ (Fig. 5C). No detectable DNA cleavage was observed in the presence of Zn2+ with any of the other noncanonical substrates. In the qualitative experiments described above, Zn2+-mediated enzyme activity appeared to be comparable with the activity in the presence of other metal ions. We resorted to kinetic analysis to obtain quantitative information about Zn2+ DNA cleavage. Kinetic analysis in the presence of Zn2+ revealed the turnover number (kcat) of the enzyme to be 2.12 min-1, which is comparable with that of Ca2+ (2.20 min-1), showing that Zn2+-mediated DNA cleavage is as efficient as Ca2+-dependent cleavage (Table 2) (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). The Km of the enzyme for the canonical sequence with Zn2+ (24 nm) is similar to that of Mg2+ (22 nm). The ability of Zn2+ to replace Mg2+ from the active site and induce specific cleavage suggests that it may suppress the Mg2+-mediated promiscuous activity of the enzyme. Results of Zn2+ chase experiments on Mg2+- or Mn2+-bound enzyme-DNA complex showed that the Mg2+- or Mn2+-mediated promiscuous activity was completely suppressed in the presence of 100 μm Zn2+ (Fig. 5D). The ability of different metal ions to bind the active site indicates the plasticity of the active site. Previous studies revealed that R.KpnI utilizes the HNH motif in its reaction mechanism for Mg2+/Mn2+/Ca2+-mediated DNA cleavage. Residues Asp148, His149, and Gln175 together form the active site and are essential for Mg2+ binding and catalysis (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar). The active site mutant enzymes D148G, H149L, and Q175E were analyzed for Zn2+-mediated DNA cleavage (supplemental Fig. 1). Zn2+-mediated cleavage also relies on the same catalytic motif, indicating the inherent flexibility of the R.KpnI active site HNH motif to accommodate Mg2+, Mn2+, Ca2+, and Zn2+. Replacement of the other metal ions from the active site by Zn2+ and retention of the catalytic activity with kinetic constants comparable with those of Mg2+, Mn2+, and Ca2+ indicate that Zn2+ is a cofactor for R.KpnI.TABLE 2Kinetic parameters of R.Kpnl with different divalent metal ionsMetal ionsKmKcatKcat/Kmmmin−1m−1 s−1Zn2+24 ± 1.6 × 10−92.12 ± 0.161.1 ± 0.4 × 106Mg2+aKinetic constants obtained from Saravanan et al. (22).22 ± 0.8 × 10−94.32 ± 0.123.2 ± 0.3 × 106Mn2+aKinetic constants obtained from Saravanan et al. (22).21 ± 1.3 × 10−94.62 ± 0.183.6 ± 0.4 × 106Ca2+aKinetic constants obtained from Saravanan et al. (22).36 ± 1.6 × 10−92.20 ± 0.181.0 ± 0.1 × 106a Kinetic constants obtained from Saravanan et al. (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). Open table in a new tab Zinc finger proteins are involved in fundamental cellular processes (viz. replication, transcription, repair, translation, and programmed cell death) (7Laity J.H. Kim B.M. Wright P.E. Curr. Opin. Struct. Biol. 2001; 11: 39-46Crossref PubMed Scopus (1096) Google Scholar). Zinc finger motifs have also been discovered and implicated in maintenance of the structural architecture in a number of nucleases (9Van Roey P. Kim C.A. Fox K.M. Belfort M. Derbyshire V. EMBO J. 2001; 20: 3631-3637Crossref PubMed Scopus (62) Google Scholar, 14Vanamee E.S. Kim P. Zhu Z. Yates D. Garman E. Xu S. Aggarwal A.K. J. Mol. Biol. 2003; 334: 595-603Crossref PubMed Scopus (8) Google Scholar, 26Raaijmakers H. Kim O. Toro I. Golz S. Kemper B. Suck D. EMBO J. 1999; 18: 1447-1458Crossref PubMed Scopus (111) Google Scholar). We demonstrate that a sequence motif (119CX8CX42CX2H174) found in R.KpnI is a zinc binding motif. Based on the conserved arrangements of cysteines and/or histidines, several classes of zinc finger families (CCHH, CCCC, CCHC, and CHCC), have been characterized and shown to be involved in interactions with DNA, RNA, or other proteins (27Berg J.M. Kim H.A. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 357-371Crossref PubMed Scopus (235) Google Scholar). CCCH-type zinc fingers were identified in a number of RNA-binding proteins of eukaryotic origin (28Amann B.T. Kim M.T. Berg J.M. Biochemistry. 2003; 42: 217-221Crossref PubMed Scopus (43) Google Scholar) and also found in Mcm10 protein, which is essential for the formation of active homocomplex (29Cook C.R. Kim G. Peterson F.C. Volkman B.F. Lei M. J. Biol. Chem. 2003; 278: 36051-36058Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The arrangement of three cysteines and a histidine in the CCCH zinc finger found in R.KpnI is rare in prokaryotic proteins. The CCCH zinc fingers were identified in replication protein A homologues in different lineages of Euryarcheaota (30Lin Y. Kim J.B. Nayannor E.K. D. Chen Y.H. Cann I.K. O. J. Bacteriol. 2005; 187: 7881-7889Crossref PubMed Scopus (14) Google Scholar), RPA41 from Pyrococcus furiosus (31Komori K. Kim Y. J. Biol. Chem. 2001; 276: 25654-25660Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and 50 S ribosomal protein L36 from Thermus thermophilus (32Boysen R.I. Kim M.W. J. Pept. Res. 2001; 57: 19-28Crossref PubMed Scopus (15) Google Scholar). R.KpnI thus is a new member of the CCCH Zn2+ finger family and is the first REase to have this motif. The Zn2+ fingers in other members having this motif listed above have varied roles in protein-protein interactions, protein-nucleic acid interactions, structural integrity, and folding. From the results presented in Fig. 2, it is clear that tightly bound Zn2+ has a structural role, since it supports Mg2+-mediated promiscuous cleavage. The second Zn2+ atom, which is loosely bound to the active site, imparts catalytic function. A peculiar characteristic of R.KpnI is its highly promiscuous behavior in the presence of Mg2+ not seen with any other REase (21Chandrashekaran S. Kim M. Radha D.R. Nagaraja V. J. Biol. Chem. 2004; 279: 49736-49740Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The Mg2+ (and Mn2+)-mediated promiscuous cleavage by R.KpnI is completely suppressed by Zn2+ meanwhile, inducing the high fidelity cleavage. The architectural plasticity of the R.KpnI active site allows the binding of Mg2+, Mn2+, Ca2+, or Zn2+, which have different coordination chemistry and geometry to induce promiscuous or specific cleavage. Thus, in R.KpnI, Zn2+ has both structural and catalytic roles, together not found in any enzyme so far. The tightly bound Zn2+ at the CCCH motif imparts structural integrity for the enzyme, whereas the readily exchangeable Zn2+ at the active site induces high specificity cleavage. Finally, Zn2+ finger motifs as such appear to be extremely rare in nucleases of prokaryotic origin. Although the HNH motif is commonly found in diverse classes of nucleases, the zinc finger motifs are found only in McrA, I-PpoI, and T4 endonuclease VII belonging to the superfamily. Although the Cys4 zinc finger of T4 endonuclease VII has a structural role, the function of similar zinc finger in McrA is not known. The two Zn2+ fingers (CCCH and CCHC) in I-PpoI are also important for structural stabilization of the protein core. The catalytic and structural role for Zn2+ in R.KpnI hints at its distant origin and possibly additional yet unknown function in Klebsiella pneumoniae. We thank J. M. Bujnicki for the R.KpnI modeling and the Departments of Biochemistry and Molecular Biophysics and Solid State Chemistry Units for circular dichroism, fluorometry, and atomic absorption spectrometry, respectively. Download .pdf (.04 MB) Help with pdf files
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