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• W1963989331 abstract "The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled. The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled. Despite extensive studies on DNA replication, relatively little is known about its in vivo organization, which, in prokaryotes, occurs at the cell membrane (1Lemon K.P. Grossman A.D. Science. 1998; 282: 1516-1519Crossref PubMed Scopus (410) Google Scholar, 2Noirot-Gros M.F. Dervyn E. Wu L.J. Mervelet P. Errington J. Ehrlich S.D. Noirot P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8342-8347Crossref PubMed Scopus (156) Google Scholar, 3Cook P.R. Science. 1999; 284: 1790-1795Crossref PubMed Scopus (632) Google Scholar, 4Firshein W. Annu. Rev. Microbiol. 1989; 43: 89-120Crossref PubMed Scopus (75) Google Scholar). The well studied Bacillus subtilis phage φ29 (5Meijer W.J.J. Horcajadas J.A. Salas M. Microbiol. Mol. Biol. Rev. 2001; 65: 261-287Crossref PubMed Scopus (155) Google Scholar) is one of the few systems for which this fundamental process has been investigated. The genome of φ29 consists of a linear double-stranded DNA (dsDNA) 1The abbreviations used are: dsDNA, double strand DNA; ssDNA, single strand DNA; 2D, two-dimensional; 3D, three-dimensional; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; r.m.s.d., root mean square deviation. 1The abbreviations used are: dsDNA, double strand DNA; ssDNA, single strand DNA; 2D, two-dimensional; 3D, three-dimensional; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence; r.m.s.d., root mean square deviation. that contains a terminal protein covalently linked at each 5′-end. Initiation of φ29 DNA replication, as well as that of most other linear genomes containing a terminal protein attached to its DNA ends, occurs via a so-called protein-primed mechanism (5Meijer W.J.J. Horcajadas J.A. Salas M. Microbiol. Mol. Biol. Rev. 2001; 65: 261-287Crossref PubMed Scopus (155) Google Scholar, 6Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (343) Google Scholar, 7Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 131-176Google Scholar). The φ29 genome encodes most, if not all, proteins required for phage DNA replication making φ29 an attractive system to study membrane-associated DNA replication.The early-expressed φ29 gene 16.7, which is conserved in all φ29-related phages studied so far, encodes a well characterized membrane protein, p16.7 (130 amino acids), that plays an important role in membrane-associated φ29 DNA replication (5Meijer W.J.J. Horcajadas J.A. Salas M. Microbiol. Mol. Biol. Rev. 2001; 65: 261-287Crossref PubMed Scopus (155) Google Scholar, 8Meijer W.J.J. Lewis P.J. Errington J. Salas M. EMBO J. 2000; 19: 4182-4190Crossref PubMed Scopus (18) Google Scholar, 9Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). It contains an N-terminal transmembrane domain which is responsible for membrane localization (9Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar) (a schematic organization of protein p16.7 is shown in Fig. 1A). Analyses of a soluble variant lacking the N-terminal membrane anchor, p16.7A, revealed that it has affinity for both single-stranded (ss) and dsDNA, as well as for the φ29 terminal protein. Moreover, p16.7A, which is a dimer in solution, can form multimers, especially upon DNA binding, and multimerization is important for the mode by which it binds DNA (9Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar, 10Serna-Rico A. Salas M. Meijer W.J.J. J. Biol. Chem. 2002; 277: 6733-6742Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 11Serna-Rico A. Muñoz-Espín D. Villar L. Salas M. Meijer W.J.J. EMBO J. 2003; 22: 2297-2306Crossref PubMed Scopus (16) Google Scholar). Recently, it was shown that the dimerization and DNA-binding activities of p16.7 are confined to the C-terminal half of the protein, p16.7C (12Muñoz-Espín D. Mateu M.G. Villar L. Marina A. Salas M. Meijer W.J.J. J. Biol. Chem. 2004; 1: 1-10Google Scholar).Here, we show that p16.7C is able to form multimers in solution and that this process is enhanced in the presence of DNA. To gain insight into the multiple features and functions of p16.7C we determined its solution and crystal structures. In addition, the protein oligomers have been analyzed both by x-ray and NMR methods. The structural features of the monomer, dimer, and oligomers and their implications for DNA binding are discussed.EXPERIMENTAL PROCEDURESBacterial Strains and Growth Conditions—Escherichia coli strain JM109 (F′ traD36 lacIq (lacZ)M15 proA+B+/e14-(McrA-)(lac-proAB) thi gyrA96 (Nalr) endA1 hsdR17 (rk- mk+) relA1 supE44 recA1 (13Yanish-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11424) Google Scholar)) was used for cloning, and E. coli strain BL21(DE3)pLysS (14Studier F.W. Moffat B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4788) Google Scholar) was used for overexpression of proteins. Kanamycin was added to E. coli cultures and plates at a final concentration of 20 μg/ml.DNA Techniques—All DNA manipulations were carried out according to Sambrook et al. (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Restriction enzymes were used as indicated by the suppliers. [γ-32P]ATP (3000 Ci/mmol) was obtained from Amersham Biosciences. Plasmid DNA was isolated using a Wizard® Plus DNA purification kit (Promega, Madison, WI). DNA fragments were isolated from gels using the QIAquick gel extraction kit (Qiagen). The dideoxynucleotide chain-termination method (16Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52357) Google Scholar) with Sequenase (United States Biochemicals sequencing kit) was used for DNA sequencing.Site-directed Mutagenesis of p16.7C—Site-directed mutations in gene 16.7C were obtained by polymerase chain reaction using the QuikChange™ Site-D mutagenesis kit (Stratagene). Plasmid pET-16.7C (12Muñoz-Espín D. Mateu M.G. Villar L. Marina A. Salas M. Meijer W.J.J. J. Biol. Chem. 2004; 1: 1-10Google Scholar) was used as template DNA.Overexpression and Purification of p16.7C and Its Derivatives—Protein p16.7C and its derivatives were overexpressed and purified using a Ni2+-NTA resin column as described before (9Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar, 12Muñoz-Espín D. Mateu M.G. Villar L. Marina A. Salas M. Meijer W.J.J. J. Biol. Chem. 2004; 1: 1-10Google Scholar). Protein p16.7Cb was obtained by digestion of protein p16.7C with the thrombin endoprotease using the Thrombin Cleavage Capture Kit (Novagen, Merck Biosciences, Darmstadt, Germany) as described before (12Muñoz-Espín D. Mateu M.G. Villar L. Marina A. Salas M. Meijer W.J.J. J. Biol. Chem. 2004; 1: 1-10Google Scholar). To obtain 15N and 13C/15N-labeled p16.7C proteins, cells were grown in U-15N and U-13C/15N Bio-express medium, respectively (Cambridge Isotope Laboratories, Andover, MA).Protein Concentration—The protein (monomer) concentration was determined by UV spectrophotometry, measuring the absorbance at 280 nm. The extinction coefficients ϵ280 of p16.7C, p16.7Cb, and pE72Q were 11360 m-1·cm-1 and that of pR98W was 16800 m-1 ·cm-1.Cross-linking Assays—Cross-linking reactions using 10 μm protein and disuccinimidyl suberate as cross-linking agent were performed as described before (9Meijer W.J.J. Serna-Rico A. Salas M. Mol. Microbiol. 2001; 39: 731-746Crossref PubMed Scopus (31) Google Scholar). After cross-linking, proteins were precipitated upon the addition of 1 volume of ice-cold 20% (w/v) trichloroacetic acid and, after resuspension, analyzed by PAGE in the presence of SDS. The proteins were visualized by Coomassie Blue staining.Gel Mobility Shift Assays—Gel retarding assays were performed as described before (11Serna-Rico A. Muñoz-Espín D. Villar L. Salas M. Meijer W.J.J. EMBO J. 2003; 22: 2297-2306Crossref PubMed Scopus (16) Google Scholar).Nuclease Digestion Assays—Nuclease digestion assays were performed using end-labeled 297-bp DNA fragments corresponding to the φ29 right end genome. Labeled fragments were either used directly (dsDNA) or after heat-denaturation (ssDNA). Micrococcal nuclease digestions of the ssDNA nucleoprotein complexes were performed as described before (11Serna-Rico A. Muñoz-Espín D. Villar L. Salas M. Meijer W.J.J. EMBO J. 2003; 22: 2297-2306Crossref PubMed Scopus (16) Google Scholar). DNase I reactions contained, in 20 μl, besides the end-labeled dsDNA fragment and the indicated amount of protein, 25 mm Tris-HCl (pH 7.5) and 10 mm MgCl2. Binding reactions were incubated for 10 min at 37 °C before 0.05 unit of DNase I (Promega) was added. Digestion was allowed to proceed for 2 min at 37 °C after which the reaction was stopped upon EDTA addition to a final concentration of 20 mm. For both the micrococcal nuclease and DNase I digestions, a phenol/chloroform extraction step was performed before the DNA was precipitated with ethanol in the presence of 15 μg of linear polyacrylamide as carrier. Next, the resuspended DNA was analyzed in denaturing 6% polyacrylamide gels. Finally, gels were dried and subjected to autoradiography.Analytical Ultracentrifugation Experiments: Sedimentation Equilibrium—The experiments were performed using a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics, using an An50Ti rotor. Whole cell buoyant molecular masses (kDa) were determined by fitting a sedimentation equilibrium model for a single sedimenting solute to individual datasets with the program EQASSOC (17Minton A.P. Schuster T.M. Lave T.M. Modern Analytical Centrifugation. Birkhauser, Boston1994: 81-93Google Scholar) (supplied by Beckman).NMR Experiments—1H NMR spectra were recorded in 85:15 1H2O: 2H2O on Bruker Avance 800, Bruker AMX-600, and Varian Unity 500-MHz spectrometers. Protein concentrations for the structural analysis of the protein dimer were in the 400–800 μm range in 200 mm NaCl, 10 mm sodium phosphate buffer, and 4 mm dithiothreitol at pH 5.0. Protein assignments were obtained using a set of 2D- and 3D- and homo- and heteronuclear NMR experiments performed on the unlabeled, 15N-labeled, and 13C/15N double-labeled molecules. Thus, HNCA, HNCO, and HN(CO)CA spectra were employed for backbone assignment. The side-chain assignments were completed with 3D-HCCH-total correlation spectroscopy experiments. NOE distance restraints were obtained from 15N- or 13C-edited 3D-NOESY spectra. In addition, 2D-NOESY, 13C-selected/12C-filtered experiments were performed on a heterolabeled dimer to analyze the interprotein contacts. This protein sample was generated by mixing equimolar amounts of unlabeled and 13C/15N double-labeled protein to a global protein concentration of 100 μm in 200 mm NaCl, 1 mm dithiothreitol, 10 mm phosphate buffer and pH 5.0. The sample was incubated at 40 °C for 24 h and then concentrated for the NMR analysis.Upper limits for proton-proton distances were obtained from NOESY cross-peak intensities at three mixing times, 50, 75, and 150 ms. Cross-peaks were classified as strong, medium, and weak corresponding to upper limits of 2.5, 3.5, and 5.0 Å. The lower limit for proton/proton distances was set as the sum of the van der Waals radii of the protons. Structure calculations were performed using the program DYANA (18Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar). A set of 2180 constraints (204 interprotein) were used in the final round of calculations. The 30 best DYANA structures in terms of target function were submitted to a simulated annealing protocol with the AMBER 5.0 package with the Cornell et al. force field (19Cornell W.D. Cieplack P.C. Bayly I. Gould I.R. Merz K. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179Crossref Scopus (11443) Google Scholar).Protein oligomerization was detected by employing diffusion-ordered spectroscopy (20Johnson C.S. Prog. NMR Spectrosc. 1999; 34: 203-256Abstract Full Text Full Text PDF Scopus (1440) Google Scholar) experiments. The average diffusion coefficients of the protein were determined at two different protein concentrations (350 and 3500 μm) and two different pH values (pH 5.0 and 7.0) in 200 mm NaCl, 10 mm sodium phosphate buffer. To get structural information about the protein oligomers 2D-NOESY experiments were also carried out with these samples. In addition, 13C-HSQC and 15N-HSQC spectra were recorded at high and low protein concentration. The high protein concentration sample was prepared by mixing unlabeled with 13C/15N double-labeled protein (4:1 ratio) to a final protein concentration of 3500 μm. Finally 2D-NOESY, 13C-selected/12C-filtered experiments were performed on this sample to analyze the interprotein contacts responsible for oligomerization.Protein Crystallization and Data Collection—Crystallization experiments were carried out with a p16.7C solution at 10 mg/ml containing 50 mm phosphate buffer, pH 7.5, 200 mm NaCl. Protein crystallization was achieved by dialysis of the protein solution against a solution containing 5 mm phosphate buffer, pH 7.5, 75 mm NaCl. Prismatic crystals (0.05 × 0.05 × 0.2 mm) appear after 2 or 3 days of incubation at 4 °C. Crystals were successively transferred to a series of solutions with increased percentage of glycerol (from 5% to 35%) and mounted in a fiber loop and frozen at 100 K in a nitrogen stream. X-ray diffraction data were collected in a charge-coupled device detector using the European Synchrotron Radiation Facility (Grenoble, France) synchrotron radiation source at wavelength 0.92 at the BM16 beam-line. Diffraction data were processed using MOSFLM (21Leslie A.G.W. Machin J.R. Papiz M.Z. Proceedings of the CCP4 Study Weekend. SERC Daresbury Laboratory, Warrington, UK1987: 39-50Google Scholar) (Table I).Table ICharacterization of the 25 NMR structures of the p16.7C dimer retained for structural analysis The average maximum violation for the upper distance constraints was 0.27 Å (average number 4.1). For the r.m.s.d. values between pairs of structures the two values correspond to superimposition of the monomeric (residues 68–125) and dimeric (residues 68a to 125a and 68b to 125b) structures.Distance constraintsNumberNOE-derived upper distance limits (Å)2180Intraresidue530Short range466Medium range670Long range514Intramonomer1976Intermonomer204r.m.s.d. from ideal geometryBonds0.0086Angle2.16Ramachandran analysis (%)Residues in most favored regions81.9Residues in additionally allowed regions18.1r.m.s.d. between pairs of structures (Å)Backbone (68–125)0.77/0.96Heavy atoms (68–125)1.46/1.56 Open table in a new tab Structure Determination and Refinement—As mentioned above, the x-ray structure of p16.7C was solved by molecular replacement using the coordinates of an ensemble of 10 preliminary NMR models (AMoRe (22Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar)). The preliminary electron density map was improved by a density average protocol performed with DM (23Cowtan K. Main P. Acta Crystallogr. Sect. D. 1998; 54: 487-493Crossref PubMed Scopus (309) Google Scholar). The averaged density map was good enough to manually refine the NMR model from residues 66a to 86b. This model was then refined using the simulated annealing routine of CNS (24Brunger A.T. Adams P.D. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar). Several cycles of restrained refinement with REF-MAC5 (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar) and iterative model building with O (26Jones T.A. Zon J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar) were carried out. Water structure was also modeled. Calculations were performed using CCP4 programs (27Bailey S. Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). The final model was refined by iterative maximum likelihood positional and translation, libration, and screw rotation displacement refinement (28Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar). The stereochemistry of the model was verified with PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Ribbon figures were produced using MOLSCRIPT (30Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and RASTER (28Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar). The accessible surface area of p16.7C dimer and protomer was calculated with the program “naccess” from the LIGPLOT package (31Wallace A.C. Laskowski R.A. Thornton J.M. Protein Eng. 1995; 8: 127-134Crossref PubMed Scopus (4253) Google Scholar).RESULTSThe Functional Domain of Protein p16.7 Can Form Multimers—Gel retardation studies demonstrated that the C-terminal half of protein p16.7 (p16.7C, containing p16.7 residues 63–130) has ss- and dsDNA-binding activities (12Muñoz-Espín D. Mateu M.G. Villar L. Marina A. Salas M. Meijer W.J.J. J. Biol. Chem. 2004; 1: 1-10Google Scholar). These experiments were performed with protein p16.7Cb, the derivative of p16.7C lacking its N-terminal (His6) tag, to exclude possible effects of this positively charged region on DNA binding. Although these experiments unequivocally demonstrated DNA-binding activity, they did not provide insight as to the ability of p16.7Cb to form multimers or the mode of DNA binding, which can be deduced by nuclease digestion analyses of the nucleoprotein complexes. Therefore, increasing amounts of p16.7Cb were incubated with 5′-labeled ss- or dsDNA probes. Next, the nucleoprotein complexes formed with ss- and dsDNA were challenged to micrococcal nuclease and DNase I digestion, respectively, after which the fragments were fractionated through denaturing polyacrylamide gels (see Fig. 1, b and c). In the absence of p16.7Cb nearly all the end-labeled ssDNA fragment was degraded into small oligonucleotides (Fig. 1b, lane 6). In the presence of p16.7Cb, in the range of 5–20 μm, however, the amount of small oligonucleotides decreased giving rise to a variety of larger ssDNA digestion products (Fig. 1b, lanes 1–3). At higher p16.7Cb concentrations, hardly any degradation products were observed (lanes 4 and 5) indicating that the ssDNA was (almost) completely protected from micrococcal nuclease attack. Together, these results confirm that p16.7Cb binds to ssDNA and, importantly, provide strong evidence that, at elevated concentrations, p16.7Cb forms multimers upon ssDNA binding leading to the generation of a continuous array of protein protecting the entire DNA fragment from micrococcal nuclease attack. Similar results were obtained when dsDNA was used (Fig. 1c). Thus, whereas a typical DNase I digestion pattern was observed in the absence or in the presence of a low amount (5 μm) of p16.7Cb (lanes 1 and 2, respectively), full protection of the dsDNA fragment was seen at higher p16.7Cb concentrations (lanes 3 and 4). Contrary to the analyses with ssDNA, though, no intermediate levels of nuclease protection were observed with dsDNA. This might indicate that multimer formation of p16.7Cb is enhanced upon binding to dsDNA, although this difference may also be due to different digestion characteristics of DNase I versus micrococcal nuclease. In vitro cross-linking carried out in the presence of dsDNA followed by Western blot analyses confirmed the ability of p16.7Cb to form multimers (Fig. 1d). Similar results were obtained using ssDNA instead of dsDNA or using p16.7C (not shown).Structure Determination of p16.7C Dimer—Analytical ultracentrifugation experiments showed that p16.7C was mainly in its dimeric form at the temperature (30–40 °C), pH (5Meijer W.J.J. Horcajadas J.A. Salas M. Microbiol. Mol. Biol. Rev. 2001; 65: 261-287Crossref PubMed Scopus (155) Google Scholar, 6Salas M. Annu. Rev. Biochem. 1991; 60: 39-71Crossref PubMed Scopus (343) Google Scholar, 7Salas M. Miller J.T. Leis J. DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 131-176Google Scholar), ionic-strength (200 mm NaCl), and concentration (0.4–0.8 mm) conditions employed for the NMR studies described below. Full backbone and side-chain assignment for p16.7C (Supplemental Table SI) was obtained using standard 2D and 3D NMR techniques on 15N- and 13C/15N-labeled samples. To distinguish intrafrom intermolecular NOEs, half-filtered NOESY experiments (Fig. 2A) were carried out on a heterolabeled dimer obtained by mixing equivalent amounts of unlabeled and 13C/15N-labeled protein. A summary of the experimental constraints employed and the characterization of the final NMR ensemble are shown in Table I.Fig. 2Solution and crystal structure of p16.7C. A, double-filtered (left) and half-filtered (right) NOESY experiments corresponding to p16.7C at 35 °C, pH 5.0, 200 mm NaCl and 10 mm sodium phosphate. Interprotein NOEs involving the methyl group of L124 are clearly observable in the half-filtered spectrum. B, backbone atoms (N, Cα, and C) of 25 superimposed NMR-derived structures of p16.7C dimer. Two different views corresponding to a 90° rotation around y-axis are shown. C, stereo ribbon representation of p16.7C crystal structure.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the course of the NMR studies, diffraction quality crystals of the protein were obtained in a dialysis membrane. These conditions were then used as a starting point for crystallographic studies. A preliminary NMR-derived ensemble of ten models was initially employed in the x-ray structural determination of p16.7C by molecular replacement at 2.9-Å resolution (see Table II and “Experimental Procedures”). The r.m.s.d. values for superimposition show that the NMR and crystal structures of p16.7C dimer are very similar (0.87 and 1.04 Å for the monomer and dimer backbone superimposition, respectively, in ordered regions; i.e. residues 68–125). The combined NMR and x-ray structures not only provide validation of one another but also give a more complete picture of the structure and dynamics of p16.7C than either structure alone.Table IIX-ray data collection and refinement statisticsData collectionSpace groupP61Unit cell (Å)a = 95.40 c = 47.57Resolution limits (Å)20–2.9I/σ (I)12.3 (3.2)aValues in parentheses are for the highest resolution shell: 2.97–2.9 ÅRmerge (%)bRmerge = ΣhklΣΣi |Ihkl–Ihkl|/ΣhklIhkl4.9 (16.7)Completeness (%)99.1 (99.8)Multiplicity7.2 (5.2)Temperature (K)120RefinementResolution range (Å)20–2.9R factor (%)23.9 (32.0)R free (%)26.5 (39.0)Number of atoms1094Number of reflections5257ModelMolecules per a.u./solvent content (%)2/65Protein/water atoms1064/19r.m.s.d. ideal bond lengths/angles (Å/deg)0.014/1.64ObservationsNo electron density for residues from 61 to 66a Values in parentheses are for the highest resolution shell: 2.97–2.9 Åb Rmerge = ΣhklΣΣi |Ihkl–Ihkl|/ΣhklIhkl Open table in a new tab Architecture of the Dimer—The solution and crystallographic 3D structures of p16.7C dimer are shown in Fig. 2 (B and C), respectively. Each polypeptide chain (green and red) contains three α helices (corresponding to p16.7 residues 72–81 (H1), 88–95 (H2), and 103–121 (H3)). Helices H1 and H2, connected by a six-residue loop (82–87; loop 1), are oriented in an antiparallel fashion with a crossing angle of ∼160°. Helix H3 is connected to H2 by a seven-residue loop (96–102; loop 2) and packs against both helix H1 and H2 with crossing angles of 54° and 139°, respectively. The C-terminal region (residues 122–128) adopts an extended structure. Finally, residues 63–66 and 129–130 are disordered (Supplemental Fig. 1B). The secondary and ternary structure of each monomer is stabilized by formation of a hydrophobic core resulting from the packing of the three helices.According to both NMR and x-ray data, p16.7C forms a symmetric dimer that corresponds to a novel six-helical fold. Indeed, none of the proteins present in the Rutgers Protein Data Bank exhibit high structural homology with p16.7C according to DALI (32Holm L. Sander C. Nucleic Acids Res. 1998; 26: 316-319Crossref PubMed Scopus (595) Google Scholar) and SCOP (33Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5567) Google Scholar). The two monomers, related by a non-crystallographic 2-fold symmetry, are held together by a combination of extensive hydrogen bonding and hydrophobic and electrostatic interactions. In the following description of the main intermolecular contacts, letters a and b will be employed to specify the polypeptide chain only when essential to avoid ambiguities. The primary dimer interface is formed by helices H3a/H3b and the extended C-terminal region of both monomers, which are oriented in an antiparallel fashion. Thus, the extended C-terminal region of each protein packs against helices H1 and H3 of the other, being involved in both hydrophobic and polar intermolecular contacts. Especially relevant for the dimer stability seems the role of Leu-124 of each monomer, which is totally buried in a hydrophobic patch formed by Tyr-79, Leu-78, and Leu-106 of the other protein monomer. In addition, there are polar intermolecular contacts between Arg-113/Lys-122, Ser-88a/Ser-88b, and Arg-85/Tyr-115. Recent studies have highlighted the influence of CH-π interactions involving proline and aromatic side chains on protein stability (34Neidigh J.W. Fesinmeyer R.M. Andersen N.H. Nat. Struct. Biol. 2002; 9: 425-430Crossref PubMed Scopus (681) Google Scholar). In p16.7C the P87a and P87b side chains, 4 Å apart at the dimer interface, pack against the Trp-116 indol ring of both polypeptides (Trp-116a and Trp-116b, Supplemental Fig. S2). This particular arrangement is likely to make a significant contribution to the stability of the complex.It has been shown that nonspecific binding of proteins to polyelectrolytes such as DNA is purely electrostatic and can be quantitatively explained in terms of competitive condensed counterion displacement from DNA by polycationic regions of the protein (35Record M.T. Lohman M.L. De Haseth P. J. Mol. Biol. 1976; 107: 145-158Crossref PubMed Scopus (929) Google Scholar). In this sense, the distribution of basic residues on the surface of a protein can provide important clues regarding nucleic acid binding sites. Thus, the surface electrostatic distribution of the p16.7C dimer was calculated to determine the most probable DNA binding site location. Fig. 3 shows that the negatively charged residues are clustered at the surface defined by helices H1/H2. In contrast, the opposite side of t" @default.
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