The Crystal Structure of the Herpes Simplex Virus 1 ssDNA-binding Protein Suggests the Structural Basis for Flexible, Cooperative Single-stranded DNA Binding

All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific, flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 Å resolution. The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain (residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees with the beaded morphology observed in electron micrographs. All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific, flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 Å resolution. The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain (residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees with the beaded morphology observed in electron micrographs. Viruses of the Herpesviridae family infect almost all vertebrates, including man, causing a variety of diseases. Of the seven viruses identified as human infectious agents, herpes simplex virus-1 (HSV-1) 1The abbreviations used are: HSV, herpes simplex virus; ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; OBP, origin-binding protein; SeMet, selenomethionine; MAD, multiwavelength anomalous diffraction; MMA, methyl mercury acetate; OB, oligonucleotide/oligosaccharide binding; HsmtSSB, human mitochondrial SSB. is the prototype of the αherpesvirus subfamily and of the family as a whole. The HSV-1 single-stranded DNA (ssDNA)-binding protein (SSB), ICP8, is a nuclear protein that, along with the six other HSV replication proteins (the viral polymerase (UL30) and its accessory factor (UL42), the trimeric helicase-primase complex (UL5-UL8-UL52), and the origin-binding protein (OBP), coded by the gene ul9), is required for viral DNA replication (1Challberg M.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9094-9098Crossref PubMed Scopus (140) Google Scholar) during lytic infection. Replication has been thought to proceed by a rolling circle mechanism (2Skaliter R. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10665-10669Crossref PubMed Scopus (57) Google Scholar) partly because the replication product is a concatamer, although the observation of highly branched replication intermediates could be explained by other mechanisms that would link recombination and replication. ICP8 is a 128-kDa multifunctional zinc metalloprotein (3Gupte S.S. Olson J.W. Ruyechan W.T. J. Biol. Chem. 1991; 266: 11413-11416Abstract Full Text PDF PubMed Google Scholar) encoded by the ul29 gene. It preferentially binds ssDNA over double-stranded DNA in a non-sequence-specific and cooperative manner (4Lee C.K. Knipe D.M. J. Virol. 1985; 54: 731-738Crossref PubMed Google Scholar). ICP8 has been reported to interact either directly or indirectly with several other viral proteins. There is evidence that it binds to the C terminus of the OBP and stimulates its helicase activity (5Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar, 6He X. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3024-3028Crossref PubMed Scopus (30) Google Scholar), that it promotes the helicase activity of the viral helicase-primase complex (UL5-UL8-UL52) (7Falkenberg M. Elias P. Lehman I.R. J. Biol. Chem. 1998; 273: 32154-32157Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and that it modulates the processivity of the viral polymerase (UL30) (8Hernandez T.R. Lehman I.R. J. Biol. Chem. 1990; 265: 11227-11232Abstract Full Text PDF PubMed Google Scholar). Before viral DNA replication commences, these proteins are thought to be co-localized with ICP8 at small punctuate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into a larger globular replication compartment (9Quinlan M.P. Chen L.B. Knipe D.M. Cell. 1984; 36: 857-868Abstract Full Text PDF PubMed Scopus (225) Google Scholar) whose location is defined by the preexisting host cell nuclear architecture, most probably at the periphery of the nuclear matrix-associated ND10 domains where the viral transactivator ICP0 and the viral input genome are believed to migrate in the early stages of infection (10Lukonis C.J. Burkham J. Weller S.K. J. Virol. 1997; 71: 4771-4781Crossref PubMed Google Scholar). ICP8 is also involved in several other events of the DNA metabolism. It can promote DNA strand transfer (11Bortner C. Hernandez T.R. Lehman I.R. Griffith J. J. Mol. Biol. 1993; 231: 241-250Crossref PubMed Scopus (60) Google Scholar), catalyze strand invasion in an ATP-independent manner (12Nimonkar A.V. Boehmer P.E. J. Biol. Chem. 2003; 278: 9678-9682Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and renature complementary DNA strands (13Dutch R.E. Lehman I.R. J. Virol. 1993; 67: 6945-6949Crossref PubMed Google Scholar), which indicates that ICP8 plays an important role in HSV genome recombination. The replication of HSV-1 DNA is also associated with a high degree of homologous recombination. Recently it was shown that ICP8 works together with alkaline nuclease (UL12), which is a 5′-3′-exonuclease, to effect strand exchange (14Reuven N.B. Staire A.E. Myers R.S. Weller S.K. J. Virol. 2003; 77: 7425-7433Crossref PubMed Scopus (91) Google Scholar). In addition to its role in DNA synthesis, ICP8 has been shown to regulate viral gene expression by repressing transcription from the parental genome (15Godowski P.J. Knipe D.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 256-260Crossref PubMed Scopus (106) Google Scholar) and stimulating late gene expression from progeny genomes (16Chen Y.M. Knipe D.M. Virology. 1996; 221: 281-290Crossref PubMed Scopus (20) Google Scholar). Genetic and biochemical analyses have failed to identify functionally independent domains within ICP8. Even the extent of the minimal DNA binding region has remained unclear. It has been placed in the C-terminal half of the protein (17Leinbach S.S. Heath L.S. Biochim. Biophys. Acta. 1989; 1008: 281-286Crossref PubMed Scopus (7) Google Scholar) or in regions spanning residues 564-1110 (18Gao M. Knipe D.M. J. Virol. 1989; 63: 5258-5267Crossref PubMed Google Scholar) or 300-849 (19Wang Y.S. Hall J.D. J. Virol. 1990; 64: 2082-2089Crossref PubMed Google Scholar). The C-terminal 60 amino acid residues were shown to account for most of the cooperative behavior in ssDNA binding (20Mapelli M. Muhleisen M. Persico G. van Der Zandt H. Tucker P.A. J. Virol. 2000; 74: 8812-8822Crossref PubMed Scopus (20) Google Scholar), possibly modulated by the two cysteines 254 and 455 (21Dudas K.C. Ruyechan W.T. J. Virol. 1998; 72: 257-265Crossref PubMed Google Scholar). It has also been shown that the C-terminal 28 amino acids contain a nuclear localization signal (22Gao M. Knipe D.M. Mol. Cell. Biol. 1992; 12: 1330-1339Crossref PubMed Scopus (45) Google Scholar), that the residues between 499 and 512 host a zinc binding motif (3Gupte S.S. Olson J.W. Ruyechan W.T. J. Biol. Chem. 1991; 266: 11413-11416Abstract Full Text PDF PubMed Google Scholar), and that the residues from 1082-1169 are also important for the stimulation of late gene expression (18Gao M. Knipe D.M. J. Virol. 1989; 63: 5258-5267Crossref PubMed Google Scholar). Here we have reported the first crystal structure of an ssDNA-binding protein of the Herpesviridae, a 60-amino acid C-terminal deletion mutant of ICP8, at 3.0 Å resolution. The structure consists of an unexpectedly large N-terminal folding unit and a small C-terminal α-helical domain, both with novel folds. In addition, it has provided insight into the likely mechanism of cooperative ssDNA binding and tempted us to speculate about the possible interaction with the origin-binding protein. The preparation and crystallization of the ICP8 protein missing the last 60 amino acids of the C terminus and with the mutations C254S and C455S (ICP8ΔCcc) have been described previously (23Mapelli M. Tucker P.A. J. Struct. Biol. 1999; 128: 219-222Crossref PubMed Scopus (7) Google Scholar). Protein Expression and Purification of Selenomethionine-ICP8ΔCcc— Selenomethionine (SeMet)-enriched ICP8ΔCcc was expressed in High 5 insect cells grown as monolayer culture. Confluent cells were infected with the same recombinant virus used for expression of the native protein in a methionine-containing Sf-900II SFM medium (Invitrogen), supplemented with 10 μg/ml penicillin-streptomycin and 2% fetal calf serum. After 30 h, cells were washed with phosphate-buffered saline solution; methionine-free IPL-41 medium (Applichem) was added for starvation. The methionine-free medium was renewed after 4 h with SeMet-containing (50 mg/l) IPL-41 medium. Cells were incubated for a further 26 h before harvesting. The purification protocol was the same used for the native protein (20Mapelli M. Muhleisen M. Persico G. van Der Zandt H. Tucker P.A. J. Virol. 2000; 74: 8812-8822Crossref PubMed Scopus (20) Google Scholar), with the only exception that all buffers were flushed with N2 and supplemented with 10 mm reducing agent (dithiothreitol or β-mercaptoethanol) to overcome a more pronounced tendency of the selenomethionine-containing protein to oxidize and aggregate. Crystallization—SeMet-ICP8ΔCcc crystals were grown at 22 °C in hanging drops by equilibration of 5 mg/ml protein in 10 mm Tris-HCl (pH 8.0), 300 mm NaBr, 20% glycerol, 10 mm dithiothreitol against 12-14% polyethylene glycol 3000 and 100 mm sodium-potassium phosphate, pH 6.3. This crystallization condition is similar to that for the native crystal growth. Within about a week, fragile, plate-like crystals (∼0.2 × 0.2 × 0.05 mm3) grew by salting in. For derivatization with methyl mercury acetate (MMA), the protein was first dialyzed against a dithiothreitol-free buffer, then against a 5 mm MMA-containing buffer at pH 7.5, and was subsequently used to set crystallization drops. Plate-like crystals appeared in a week in conditions similar to the ones used for native crystal growth. Crystals formed in space group P212121 with two molecules in the asymmetric unit. Data Collection, Structure Determination, and Refinement—Both SeMet- and MMA-containing ICP8ΔCcc crystals were cryoprotected by brief soaks in 20% glycerol buffered at pH 6.3 before cryocooling in liquid nitrogen. Multiwavelength anomalous diffraction data from crystals of SeMet-ICP8ΔCcc were collected at 100 K using synchrotron radiation at the 17-ID IMCA-CAT beamline of the Advanced Photon Source (Argonne) at three/two different wavelengths around the selenium absorption edge. A full diffraction data set was collected for the MMA derivative at 100 K, using the BW7B beam line of the European Molecular Biology Laboratory Hamburg Outstation. The diffraction data were processed using the HKL program package (24Otwinowski Z. Minor W. Carter C.W. Sweet R.M. Macromolecular Crystallography, Part A. 276. Academic Press, New York1997: 307-326Google Scholar). Data collection statistics are shown in Table I.Table ISummary of data collection and refinement statistics for ICP8CRYST-1CRYST-2CRYST-3PeakaSelenomethionine-substituted ICP8 MAD data were collected at Advanced Photon Source, beamline 17-ID IMCA-CATInflectionaSelenomethionine-substituted ICP8 MAD data were collected at Advanced Photon Source, beamline 17-ID IMCA-CATRemoteaSelenomethionine-substituted ICP8 MAD data were collected at Advanced Photon Source, beamline 17-ID IMCA-CATPeakaSelenomethionine-substituted ICP8 MAD data were collected at Advanced Photon Source, beamline 17-ID IMCA-CATRemoteaSelenomethionine-substituted ICP8 MAD data were collected at Advanced Photon Source, beamline 17-ID IMCA-CATHg-derivativebMethyl-mercury acetate-treated native ICP8 crystal data were collected at the European Molecular Biology Laboratory-Hamburg Outstation, beam line BW7A. The numbers in parentheses are for the highest resolution shellWavelength (Å)0.9794540.979680.956770.979440.956770.8467Unit cell (Å)a = 103.12a = 102.01a = 101.97a = 100.94a = 101.04a = 100.91P212121b = 147.60b = 146.13b = 146.09b = 145.61b = 145.72b = 145.37c = 170.11c = 168.39c = 168.39c = 163.26c = 163.69c = 162.03Resolution (Å)4.03.24.04.04.04.03.0Complete (%)cComplete represents (number of independent reflections)/(total theoretical number)98/(99)90/(96)99/(99)99/(99)100/(100)99/(99)98/(97)Rmerge (%)dRmerge(I) = [Σ|I(i) - 〈I(h)〉|/ΣI(i)], where I(i) is the ith observation of the intensity of the hkl reflection and 〈I〉 is the mean intensity from multiple measurements of the hkl reflection6.2/(9.4)9.3/(31.4)5.7/(12.2)5.6/(11.3)7.4/(13.1)7.0/(14.6)9.0/(30.3)I/σ(I)18/(13)15/(5)15/(7)16/(8)16/(9)13/(6)10/(3)Refinement statistics of CRYST-3 Resolution (Å)20-3.0 (Å)Number of nonhydrogen atoms Reflections in working set46078Protein15691 Reflections in test set1185Water95 Rfree (%)eRfree is calculated over reflections in a test set not included in atomic refinement28.6Number of metal sites (Zn, Hg)2, 6 Rcryst (%)fRcryst(F) = Σh||Fobs(h)| - |Fcalc(h)||/Σh|Fobs(h)|, where |Fobs(h)| and |Fcalc(h)| are the observed and calculated structure factor amplitudes for the hkl reflection23.5Residue distribution in Ramachandran plotgRef. 33Most favorable region (%)85.6Allowed region (%)13.3a Selenomethionine-substituted ICP8 MAD data were collected at Advanced Photon Source, beamline 17-ID IMCA-CATb Methyl-mercury acetate-treated native ICP8 crystal data were collected at the European Molecular Biology Laboratory-Hamburg Outstation, beam line BW7A. The numbers in parentheses are for the highest resolution shellc Complete represents (number of independent reflections)/(total theoretical number)d Rmerge(I) = [Σ|I(i) - 〈I(h)〉|/ΣI(i)], where I(i) is the ith observation of the intensity of the hkl reflection and 〈I〉 is the mean intensity from multiple measurements of the hkl reflectione Rfree is calculated over reflections in a test set not included in atomic refinementf Rcryst(F) = Σh||Fobs(h)| - |Fcalc(h)||/Σh|Fobs(h)|, where |Fobs(h)| and |Fcalc(h)| are the observed and calculated structure factor amplitudes for the hkl reflectiong Ref. 33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar Open table in a new tab The structure was solved by the MAD method (25Hendrickson W.A. Science. 1991; 254: 51-58Crossref PubMed Scopus (1016) Google Scholar). Initially from a first SeMet-containing crystal (CRYST-1), FA values were obtained using XPREP (Bruker-AXS Inc.) to 4.0 Å, enabling the selenium substructure to be solved (50 of 56 seleniums) using the program SHELXD (26Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1578) Google Scholar). Phases were then obtained to 4.0 Å from the two wavelength MAD data. The phases were extended to 3.2 Å by using density modification procedures and 2-fold non-crystallographic symmetry averaging (27Cowtan K.D. Main P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1996; 52: 43-48Crossref PubMed Scopus (288) Google Scholar). 55% of the model was built using a semiautomatic procedure with the programs MAID (28Levitt D.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1013-1019Crossref PubMed Scopus (117) Google Scholar), RESOLVE (29Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1937-1940Crossref PubMed Scopus (283) Google Scholar), and O (30Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar). Later, phases were extended to 3.0 Å using data from another crystal (CRYST-3, see Table I) by applying multiple crystal averaging (31Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19767) Google Scholar). The resultant phases allowed the Se substructure of CRYST-2 to be determined using an anomalous difference Fourier at 4.0 Å. Then single isomorphous replacement with anomalous scattering was used to calculate phases, and phase combination was performed to 4.0 Å with the phases generated from multiple crystal averaging. Finally, phases were extended to 3.0 Å using density modification and 2-fold non-crystallographic symmetry averaging. At this stage, the quality of the map improved significantly. Model building was continued in a similar manner to that described above, and 70% of the model could be built. Refinement of the structure was performed using simulated annealing, followed by positional and restrained B-factor refinement as implemented in CNS (32Brünger A.V. Adams P.P. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16966) Google Scholar). As the model became more complete, a new mask was calculated and used in the multiple crystal averaging and phase combination. Density modification and 2-fold non-crystallographic symmetry averaging were repeated, followed by the semiautomatic procedure for model building. The model produced in this way was nearly complete except for some missing loops, and there was interpretable density for 90% of the residues. In the final stage, refinement was continued using non-crystallographic symmetry restraints and a bulk solvent correction in the program CNS (32Brünger A.V. Adams P.P. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16966) Google Scholar). The refinement was monitored using the free R-factor calculated with 10% of observed reflections. The refinement statistics for CRYST-3 (which, although a mercury derivative, were the best 3.0 Å data) are shown in Table I. Of 1136 residues, 107 residues in chain A and 105 in chain B are not visible in the electron density and are probably disordered. The major disordered loops are located at the interface of the neck and head. The rest of the disordered loops are situated at different parts of the shoulder region and are shown as dotted lines in Fig. 1. Overall geometric quality of the model was assessed using PRO-CHECK (33Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). 86% of the amino acid residues of ICP8 were found in the most favorable region of the Ramachandran plot, with the remaining residues (apart from Thr908) in the additional and generously allowed regions. All figures were produced using MOLSCRIPT (34Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), PyMol (35De Lano W.L. Pymol User Manual. DeLano Scientific, San Carlos, CA2002Google Scholar), and RASTER3D (36Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2857) Google Scholar). Modeling of ssDNA—The structure of the EcoSSB-ssDNA complex, where two monomers of EcoSSB cover 26 nucleotides, (37Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (361) Google Scholar) was used for the modeling of ssDNA onto the neck region of ICP8. One of the two monomers was superimposed with a monomer of human mitochondrial SSB (HsmtSSB) (38Yang C. Curth U. Urbanke C. Kang C. Nat. Struct. Biol. 1997; 4: 153-157Crossref PubMed Scopus (150) Google Scholar) with a root mean square deviation of 1.6 Å on the 97-Cα target pair, and then the monomer of HsmtSSB was overlaid on the neck region of ICP8. In this way the relative orientation of ssDNA was modeled onto the complete structure of ICP8 with the ssDNA covering the neck region of ICP8. The two independent molecules that form the protein chain have different spatial arrangements of the C- and N-terminal domains; however, the distance over the disordered region (1038-1048) is approximately the same (16.1 and 19.2 Å). The relative orientation of ssDNA was created for each monomer of the proposed protein chain formed by applying non-crystallographic symmetry. Visual inspection using computer graphics allowed the nucleotides between two non-crystallographic symmetry-related monomers (chains A and B) and between two crystallographically related monomers (chain B and symmetry mate of chain A) to be added while avoiding clashes with the protein chain. Again the crystallographic symmetry was applied to the newly built nucleotides. In this way, it was possible to join the ssDNA in a continuous chain. In the model the continuous chain of ssDNA contains 98 nucleotides covered by 7 monomers and the distance between 5′- and 3′-ends is 350 Å. The coordinates of the model are available from the authors upon request, and a more detailed illustration is included in the supplemental material. Crystallization and Structure Determination—The structure and function of a number of other prokaryotic and eukaryotic SSBs have been described (39Agrawal V. Kishan K.V. Curr. Protein Pept. Sc. 2003; 4: 195-206Crossref PubMed Scopus (43) Google Scholar), but ICP8 is much larger (128 kDa) relative to other SSBs. For example, the monomers of bacterial SSBs are typically ∼20 kDa, and although the heterotrimeric eukaryotic SSB, RepA, is ∼116 kDa, this is believed to contain more than one DNA binding region. Crystallization studies of ICP8 have been reported earlier (23Mapelli M. Tucker P.A. J. Struct. Biol. 1999; 128: 219-222Crossref PubMed Scopus (7) Google Scholar), and it was shown that crystals of full-length ICP8 diffracted too poorly to be useful. A mutant, ICP8ΔCcc, with the C-terminal 60 residues deleted and two point mutations (C254S and C455S) has been crystallized under similar conditions to the full-length ICP8 and diffracted to at least 3 Å resolution. The mutant has been shown to bind ssDNA with much reduced cooperativity (22Gao M. Knipe D.M. Mol. Cell. Biol. 1992; 12: 1330-1339Crossref PubMed Scopus (45) Google Scholar). The structure was solved by MAD and single isomorphous replacement with anomalous scattering methods using SeMet-substituted ICP8. The SeMet crystals formed in space group P212121 with two molecules in the asymmetric unit and diffracted to 3.2 Å resolution. The model (residues 9-1129 with the disordered regions described below) was refined to a crystallographic R value of 23.5% (Rfree = 28.6%) using data from 20.0 to 3.0 Å resolution (Table I). Overall Structure—The structure of ICP8 (9-1129) (Fig. 1) is composed of a large N-terminal domain (9-1038) and a smaller α-helical C-terminal domain (1049-1129). The first 8 residues and the last 7 residues of the construct are not visible in the electron density and are presumed to be disordered. The N-terminal domain can be described as consisting of head, neck, and shoulder regions. The head consists of the eight helices α14, α15, α16, α21, α22, α23, α24, and α25 (Fig. 1B). The front side of the neck region consists of a five-stranded β-sheet (β16, β17, β23, β26, and β27) and two helices (α17 and α27), whereas the back side is a three-stranded β-sheet (β24, β25, and β28) (Fig. 1). The shoulder part of the N-terminal domain contains an α-helical and β-sheet region. The head, neck, and shoulders are interconnected in such a way that their individual structural folds are not formed by contiguous polypeptide chains. From the N terminus, the polypeptide chain forms a first helical region in the head and then one of the two β-sheet regions belonging to the neck. The strands β16 and β17 in the neck lead to strands β18-β22 in the shoulders before returning to strands β23-β26 in the neck. The strands β18-β22 are involved in interaction with residues in other strands from the N terminus (see Fig. 1A). This explains why limited proteolysis experiments have never yielded either soluble or functionally active fragments (20Mapelli M. Muhleisen M. Persico G. van Der Zandt H. Tucker P.A. J. Virol. 2000; 74: 8812-8822Crossref PubMed Scopus (20) Google Scholar) and why so many mutant proteins have proven to be insoluble (see, for example, Ref. 18Gao M. Knipe D.M. J. Virol. 1989; 63: 5258-5267Crossref PubMed Google Scholar). The C-terminal domain (1049-1129) is entirely helical (α28, α29, α30, α31, and α32) and is connected to the N-terminal domain ∼17 Å away by a disordered linker (residues 1038-1049) (Figs. 1 and 2). DNA Binding Region—No structurally related protein can be retrieved from the DALI (40Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3563) Google Scholar) or SSM (www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver) servers using the whole ICP8 molecule or the individual subdomains as search models. Although no structural homology is detectable for any of the ICP8 regions (Fig. 1), the front side of the neck region shows some structural resemblance to the oligonucleotide/oligosaccharide binding (OB) fold (41Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (774) Google Scholar), which is responsible for ssDNA binding in all SSBs so far described with the exception of the adenoviral SSB (42Tucker P.A. Tsernoglou D. Tucker A.D. Coenjaerts F.E. van der Leenders H. Vliet P.C. EMBO J. 1994; 13: 2994-3002Crossref PubMed Scopus (67) Google Scholar). The topology is different (39Agrawal V. Kishan K.V. Curr. Protein Pept. Sc. 2003; 4: 195-206Crossref PubMed Scopus (43) Google Scholar), but the principle is the same, namely a crossed β-sheet with disordered connecting loops containing conserved basic and aromatic residues. The direction of each β-strand of the neck region that resembles the OB-fold is similar to that of HsmtSSB (38Yang C. Curth U. Urbanke C. Kang C. Nat. Struct. Biol. 1997; 4: 153-157Crossref PubMed Scopus (150) Google Scholar) (Fig. 3A). The proposed DNA binding region on the front side of the neck (Fig. 3A) contains elements of the sequence between amino acids 530 and 1028, similar to the boundaries suggested by Gao and Knipe (18Gao M. Knipe D.M. J. Virol. 1989; 63: 5258-5267Crossref PubMed Google Scholar). Limited proteolytic analysis studies had suggested that the putative boundaries of the minimal DNA binding region are between residues 300 and 849 (19Wang Y.S. Hall J.D. J. Virol. 1990; 64: 2082-2089Crossref PubMed Google Scholar). More recent evidence, based on ICP8 photo-affinity labeling with oligonucleotides, indicated a slightly different region, namely between residues 386 and 902 (43White E.J. Boehmer P.E. Biochem. Biophys. Res. Commun. 1999; 264: 493-497Crossref PubMed Scopus (8) Google Scholar). There are a number of aromatic and positively charged residues from the front side of the neck that are exposed to the surface or lie in the disordered loops that are relatively well conserved across the Herpesviridae. These are Tyr543, Asn551, Arg772, Lys774, Arg776, Tyr988, Phe998, and Asn1002 (Fig. 4), which we believe are involved in ssDNA binding either by base stacking or electrostatic interactions.Fig. 4Structure-based sequence alignment of the ICP8 of Herpesviridae from three subfamilies. Representatives from three genus of the Alphaherpes virus subfamily (Simplexvirus, Varicellavirus, Marek's disease-like viruses), three genus from the Betaherpes virus subfamily (Roseolovirus, Cytomegalovirus, Muromegalovirus), and two genus from the gammaherpes virus subfamily (Rhadinovirus, Lymphocryptovirus) are used in the sequence alignment. The Swiss-Prot codes of ICP8 orthologues from these sources are P04296, Q89549, Q9E6P0, O56282, P17147, P30672, O36360 and P03227, respectively. Horizontal cylinders above the sequences indicate α-helices (labeled α1-α32). Horizontal arrows indicate β-strands (labeled β1-β28). The secondary structure elements are colored red for the head, blue and orange for the shoulder, yellow and gray for the neck region of the N-terminal domain (a similar color code is used in Fig. 1A), purple for the C-terminal helical domain, and light green for the zinc binding loop, including two helices (α12 and α13) that are involved in interaction with part of the N and C termini. Three cysteines and a histidine involved in binding to zinc are shown by a bar above the corresponding residues. The dotted lines indicate regions that are disordered inthe crystal structure. The dashed line indicates that the region was absent in the construct. The triangle above 3 residues in the C-terminal region (last 60 residues) indicates the region encompassing the FNF motif. The star sign above two cysteines shows mutation to serine in the structure presented here.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Role of Zinc Binding Region—ICP8 is a zinc metalloprotein containing one zinc atom/molecule (3Gupte S.S. Olson J.W. Ruyechan W.T. J. Biol. Chem. 1991; 266: 11413-11416Ab