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• W2010707114 abstract "KorB is a regulatory protein encoded by the conjugative plasmid RP4 and a member of the ParB family of bacterial partitioning proteins. The protein regulates the expression of plasmid genes whose products are involved in replication, transfer, and stable inheritance of RP4 by binding to palindromic 13-bp DNA sequences (5′-TTTAGC(G/C)GCTAAA-3′) present 12 times in the 60-kb plasmid. Here we report the crystal structure of KorB-C, the C-terminal domain of KorB comprising residues 297–358. The structure of KorB-C was solved in two crystal forms. Quite unexpectedly, we find that KorB-C shows a fold closely resembling the Src homology 3 (SH3) domain, a fold well known from proteins involved in eukaryotic signal transduction. From the arrangement of molecules in the asymmetric unit, it is concluded that two molecules form a functionally relevant dimer. The detailed analysis of the dimer interface and a chemical cross-linking study suggest that the C-terminal domain is responsible for stabilizing the dimeric form of KorB in solution to facilitate binding to the palindromic operator sequence. The KorB-C crystal structure extends the range of protein-protein interactions known to be promoted by SH3 and SH3-like domains. KorB is a regulatory protein encoded by the conjugative plasmid RP4 and a member of the ParB family of bacterial partitioning proteins. The protein regulates the expression of plasmid genes whose products are involved in replication, transfer, and stable inheritance of RP4 by binding to palindromic 13-bp DNA sequences (5′-TTTAGC(G/C)GCTAAA-3′) present 12 times in the 60-kb plasmid. Here we report the crystal structure of KorB-C, the C-terminal domain of KorB comprising residues 297–358. The structure of KorB-C was solved in two crystal forms. Quite unexpectedly, we find that KorB-C shows a fold closely resembling the Src homology 3 (SH3) domain, a fold well known from proteins involved in eukaryotic signal transduction. From the arrangement of molecules in the asymmetric unit, it is concluded that two molecules form a functionally relevant dimer. The detailed analysis of the dimer interface and a chemical cross-linking study suggest that the C-terminal domain is responsible for stabilizing the dimeric form of KorB in solution to facilitate binding to the palindromic operator sequence. The KorB-C crystal structure extends the range of protein-protein interactions known to be promoted by SH3 and SH3-like domains. C-terminal domain (amino acids 297–358) of KorB protein N-terminal domain (amino acids 1–294) of KorB protein root mean square Src homology 3 KorB is encoded on the central control region of the plasmid RP4. This plasmid is a member of Escherichia coliincompatibility group P (IncP-1α), and it is indistinguishable from plasmids R18, R68, RK2, and RP1. The IncP-1α plasmids RP4/RP1/RK2 were the first to be studied in great molecular detail (1Pansegrau W. Lanka E. Barth P.T. Figurski D.H. Guiney D.G. Haas D. Helinski D.R. Schwab H. Stanisich V.A. Thomas C.M. J. Mol. Biol. 1994; 239: 623-663Crossref PubMed Scopus (429) Google Scholar). RP4 is a self-transmissible resistance plasmid of about 60 kb. Broad host-range IncP-1α plasmids are of particular interest due to their promiscuity, which is exhibited as their capacity to transfer and stably maintain themselves in a wide variety of Gram-negative bacterial species (2Thomas C.M. Smith C.A. Nucleic Acids Res. 1986; 14: 4453-4469Crossref PubMed Scopus (52) Google Scholar). A major role in the ability of these plasmids to survive is played by plasmid regulators that control and coordinate replication, transfer, and partitioning functions (3Jagura-Burdzy G. Thomas C.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10571-10575Crossref PubMed Scopus (40) Google Scholar, 4Jagura-Burdzy G. Thomas C.M. J. Mol. Biol. 1997; 265: 507-518Crossref PubMed Scopus (25) Google Scholar, 5Motallebi-Veshareh M. Balzer D. Lanka E. Jagura-Burdzy G. Thomas C.M. Mol. Microbiol. 1992; 6: 907-920Crossref PubMed Scopus (62) Google Scholar, 6Zatyka M. Jagura-Burdzy G. Thomas C.M. Microbiology. 1997; 140: 2981-2990Crossref Scopus (40) Google Scholar, 7Zatyka M. Jagura-Burdzy G. Thomas C.M. J. Bacteriol. 1997; 179: 7201-7209Crossref PubMed Google Scholar). These regulators are KorA, KorB, KorC, and TrbA. KorB plays a direct role in the partitioning of the plasmid, acting together with another protein. KorB also functions as a transcriptional repressor of RP4 genes. It is a member of the ParB family of proteins that are involved in genome partitioning and encoded on plasmids and bacterial chromosomes (8Lobocka M. Yarmolinsky M. J. Mol. Biol. 1996; 259: 366-382Crossref PubMed Scopus (68) Google Scholar, 9Motallebi-Veshareh M. Rouch D. Thomas C.M. Mol. Microbiol. 1990; 4: 1455-1463Crossref PubMed Scopus (167) Google Scholar, 10Williams D.R. Thomas C.M. J. Gen. Microbiol. 1992; 138: 1-16Crossref PubMed Scopus (160) Google Scholar, 11Williams D.R. Macartney D.P. Thomas C.M. Microbiology. 1998; 144: 3369-3378Crossref PubMed Scopus (63) Google Scholar). Purified KorB exists as a dimer in solution (12Balzer D. Ziegelin G. Pansegrau W. Kruft V. Lanka E. Nucleic Acids Res. 1992; 20: 1851-1858Crossref PubMed Scopus (125) Google Scholar), and even formation of a homo-oligomer was described (13Williams D.R. Motallebi-Veshareh M. Thomas C.M. Nucleic Acids Res. 1993; 21: 1141-1148Crossref PubMed Scopus (51) Google Scholar). KorB has a size of 358 amino acids (39,011 Da) with an abundance of about 2000 molecules/cell. Despite its negative charge at neutral pH, KorB recognizes and binds specifically to the operator sequence, OB (5′-TTTAGC(G/C)GCTAAA-3′), which occurs 12 times on the RP4 genome. The positions of these 12 operator sites (measuring from the middle of the OB site) relative to RP4 promoters can be classified as follows. Class I sites are 39/40 bp upstream of a transcription start point, class II sites are further upstream or downstream of promoters but within 80–190 bp of a transcription start site, and class III sites are more than 1 kb away from any known promoter (14Jagura-Burdzy G. Macartney D.P. Zatyka M. Cunliffe L. Cooke D. Huggins C. Westblade L. Khanim F. Thomas C.M. Mol. Microbiol. 1999; 32: 519-532Crossref PubMed Scopus (38) Google Scholar). KorB can repress the RP4 promoters carrying class I or class II OB sites (4Jagura-Burdzy G. Thomas C.M. J. Mol. Biol. 1997; 265: 507-518Crossref PubMed Scopus (25) Google Scholar, 5Motallebi-Veshareh M. Balzer D. Lanka E. Jagura-Burdzy G. Thomas C.M. Mol. Microbiol. 1992; 6: 907-920Crossref PubMed Scopus (62) Google Scholar, 14Jagura-Burdzy G. Macartney D.P. Zatyka M. Cunliffe L. Cooke D. Huggins C. Westblade L. Khanim F. Thomas C.M. Mol. Microbiol. 1999; 32: 519-532Crossref PubMed Scopus (38) Google Scholar, 15Macartney D.P. Williams D.R. Stafford T. Thomas C.M. Microbiology. 1997; 143: 2167-2177Crossref PubMed Scopus (43) Google Scholar, 16Thomson V.J. Jovanovic O.S. Pohlman R.F. Chang C.H. Figurski D.H. J. Bacteriol. 1993; 175: 2423-2435Crossref PubMed Google Scholar), but its role in the regions where class III operators occur has not been elucidated. However, the conservation of these sites on the IncP-1 relative of RP4, plasmid R751, whose complete sequence was compiled (17Thorsted P.B. Macartney D.P. Akhtar P. Haines A.S. Ali N. Davidson P. Stafford T. Pocklington M.J. Pansegrau W. Wilkins B.M. Lanka E. Thomas C.M. J. Mol. Biol. 1998; 282: 969-990Crossref PubMed Scopus (183) Google Scholar), suggests that even class III OB sites play an important role in the expression of the genome. In conjunction with KorA, KorB represses the transcription of the kilA,trfA, and korAB operons. It is also involved in the negative control of kilB operons. KorA and KorB act cooperatively in transcriptional repression. Detailed studies on the binding of KorB to the 12 operators present in RP4 showed that they fall into three groups according to the binding strength of KorB. The highest affinity site is OB10, which occurs in the promoter transcribing genes for replication,trfAp. Purified IncC1 enhanced KorB binding to all OB sites except OB3, a site involved in partitioning (18Kostelidou K. Thomas C.M. J. Mol. Biol. 2000; 295: 411-422Crossref PubMed Scopus (31) Google Scholar). The 5-bp sequences flanking the 13-mer OB site were found to affect KorB binding and IncC1 stimulatory activity. Flanking sequences on one side only were sufficient to specify the difference between OB10 and OB3. These differences also eliminated potentiation by IncC1. It was suggested that KorB contacts OB flanking sequences and that IncC1 may alter the conformation of multimeric KorB so that it is better able to make these contacts, thus stabilizing the complexes once formed. To elucidate the structural basis of KorB's DNA-binding properties and the interaction with other proteins, a crystallographic analysis was initiated. Here we report the crystal structure of the C-terminal domain, KorB-C,1 consisting of 62 amino acids. This is the first crystal structure of a protein encoded by the RP4 plasmid. We have solved the structure in two different crystal forms observing a SH3-like monomer fold and closely similar dimeric arrangements in three crystallographically independent copies. Based on the structure, we argue that the C-terminal domain is mainly responsible for stabilizing the dimeric form of KorB, thus enhancing specific operator binding by the repressor. korB was overexpressed in E. coli, and the protein was purified as described by Balzer et al. (12Balzer D. Ziegelin G. Pansegrau W. Kruft V. Lanka E. Nucleic Acids Res. 1992; 20: 1851-1858Crossref PubMed Scopus (125) Google Scholar). The highly soluble protein (>100 mg ml−1) was subjected to crystallization experiments. After several months, a crystal with a hexagonal unit cell of a = 51.8 Å, c = 88.3 Å was obtained. Even under the most favorable assumptions of one KorB molecule per asymmetric unit and point symmetry 6 of the crystal, this unit cell is too small to accommodate intact KorB, yielding an unlikely Matthews coefficient (19Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7927) Google Scholar), VM, of 0.88 Å3 Da−1. Therefore, the crystal was dissolved and analyzed by mass spectrometry and N-terminal sequencing. The result was that the crystal contained only a C-terminal KorB fragment extending from residue 297 to the end. The corresponding 3′-terminal fragment of korB was molecularly cloned, and the product KorB-C was overproduced in E. coli in the same way as the intact protein (12Balzer D. Ziegelin G. Pansegrau W. Kruft V. Lanka E. Nucleic Acids Res. 1992; 20: 1851-1858Crossref PubMed Scopus (125) Google Scholar). KorB-C was purified in a four-step procedure. In an initial step, the cell extract was loaded onto a DEAE-Sephacel (Amersham Biosciences, Inc.) column and eluted with a linear NaCl gradient. The pooled fractions were put on a heparin column (Amersham Biosciences HiTrap Heparin HP) and eluted with a 0–0.25 m NaCl gradient. The collected fractions were purified again with MonoQ (Amersham Biosciences HR 5/5). Finally, KorB-C was purified to homogeneity by size exclusion chromatography (Amersham Biosciences Superdex G75). A 5′-terminal fragment ofkorB was cloned analogously, and the product, KorB-N (residues 1–294 of KorB), was overproduced in E. coli. For crystallization, KorB-C was dialyzed against 20 mmTris-HCl, pH 7.6, and 50 mm NaCl and concentrated. Different crystal forms were obtained under several conditions in hanging drop vapor diffusion setups, sometimes even in the same drop. Crystals used for data collection were grown under the following conditions. Hexagonal crystal (P65) with a= 51.8 Å, c = 88.3 Å grew from 30% polyethylene glycol 4000 and 200 mm ammonium acetate in 100 mm sodium acetate, pH 4.6. These crystal parameters are the same as those found for the KorB-C crystals obtained from degraded KorB. A second crystal form was obtained from 36% polyethylene glycol 4000 and 200 mm sodium chloride in 100 mmsodium acetate, pH 4.6. These crystals belong to space group P212121 with a = 42.1 Å, b = 82.1 Å, c = 87.9 Å. For data collection, the crystals were flash-frozen directly from the crystallization droplet. Heavy atom derivatives were prepared by soaking orthorhombic crystals with platinum tetrachloride andcis-diamminplatinum chloride until the crystals displayed the color of the heavy atom salt solution. X-ray diffraction data were collected in-house and at beamline X31 of EMBL/DESY in Hamburg and processed with DENZO/HKL (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The structure was solved in the orthorhombic space group using a MIRAS approach based on three diffraction data sets collected for two platinum derivatives (TableI). One derivative data set was collected at the Pt LIII peak wavelength as established from a fluorescence scan to optimize the anomalous signal. Four heavy atom sites present in each derivative but differing in relative occupancy and anomalous contribution were identified by SOLVE (21Terwilliger T.C. Berendzen J. Acta Crystallogr. Sec. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The phases were refined with MLPHARE, and the quality of the density was improved by solvent averaging using DM as implemented in the CCP4 package (22Collaborative Computational Project 4Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). A first inspection of the electron density revealed the noncrystallographic symmetry present in the asymmetric unit and permitted the subsequent phase improvement by noncrystallographic symmetry averaging in DM. This yielded an easily interpretable electron density map. An atomic model of KorB-C was built in O (23Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and initially refined in XPLOR 3.7 (24Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar). Finally, the structure was refined to convergence in REFMAC (25Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sec. D. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) using isotropic B values for all nonhydrogen atoms. Noncrystallographic symmetry restraints were gradually decreased and completely removed in the last refinement cycles. All solvent peaks were modeled as fully occupied water oxygens. Alternative conformations were modeled for a number of side chains and occupancies adjusted to yield similar B values for both conformers.Table ICrystallographic data and phasing statisticsNativeDerivativeOrthorhombicHexagonalPtCl4PtCl4Cis-platin(II) diamminchlorideSpace groupP212121P65P212121P212121P212121Wavelength (Å)1.07211.54181.07211.54181.5418Unit cell (Å)a = 42.13a = 51.68a = 43.05a = 42.05a = 43.04b = 82.15c = 87.42b = 81.39b = 81.29b = 81.79c = 87.86c = 87.58c = 87.65c = 88.01Resolution (Å)22–1.7722–2.1540–2.5536–2.5036–2.75Unique reflections32,1607,1579,81210,8978,515Completeness1-aValues for whole dataset/highest resolution bin.(%)98.9/95.799.0/87.994.9/87.998.3/89.998.7/86.5Redundancy1-aValues for whole dataset/highest resolution bin.3.4/3.33.4/2.54.0/3.93.8/3.52.7/2.3I/ς(I)1-aValues for whole dataset/highest resolution bin.19.5/3.88.5/3.94.0/1.79.9/2.89.7/2.0〈B〉 from Wilson plot19.525.242.645.643.7R(sym)1-bR(sym) = ∑i∑j‖〈Ii〉 − Ii,j‖/∑i∑jIi,j, where Ii,j are the measurements of contribution to the mean reflection intensity 〈Ii〉.(%)4.4/20.37.1/19.510.95.710.6R(iso)1-cR(iso), phasing power, R(Cullis), and figure or merit (f.o.m.) are according to MLPHARE (21).(%)46.245.620.7No. of sites444Phasing power1-cR(iso), phasing power, R(Cullis), and figure or merit (f.o.m.) are according to MLPHARE (21).acen./cen.1.76/1.222.04/1.461.20/0.98R(Cullis)1-cR(iso), phasing power, R(Cullis), and figure or merit (f.o.m.) are according to MLPHARE (21).acen./cen./anom.0.69/0.73/0.830.64/0.68/0.870.83/0.77/0.98f.o.m.1-cR(iso), phasing power, R(Cullis), and figure or merit (f.o.m.) are according to MLPHARE (21).0.51601-a Values for whole dataset/highest resolution bin.1-b R(sym) = ∑i∑j‖〈Ii〉 − Ii,j‖/∑i∑jIi,j, where Ii,j are the measurements of contribution to the mean reflection intensity 〈Ii〉.1-c R(iso), phasing power, R(Cullis), and figure or merit (f.o.m.) are according to MLPHARE (21Terwilliger T.C. Berendzen J. Acta Crystallogr. Sec. D. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Open table in a new tab The structure of KorB-C in the hexagonal space group was solved by molecular replacement with AMORE (26Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) using a protein dimer from the orthorhombic KorB-C structure as a model. The structure of hexagonal KorB-C was refined essentially as the orthorhombic structure. KorB-C protein (15 μm) was treated with increasing concentrations of glutardialdehyde in a buffer consisting of 20 mm NaH2PO4 (pH 7.0), 100 mm NaCl, 1 mm dithiothreitol and 5% glycerol. After a 1-h incubation at 37 °C, the reaction was stopped by adding 1 m Tris-HCl, pH 7.6, to a concentration of 100 mm. The products were reduced, heat-denatured, and electrophoresed on a 17.5% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS. Complex formation of KorB proteins with OB DNA was assayed by polyacrylamide gel electrophoresis. A DNA digest containing equimolar amounts of fragments was incubated for 30 min at 37 °C with 3 or 9 pmol of KorB or with 2.5–45 pmol of either KorB-C or KorB-N and electrophoresed at 8 V cm−1 on a nondenaturing 3.5% PAGE containing 40 mm Tris-acetic acid, pH 7.9, 20 mm sodium acetate, 2 mm EDTA according to Balzer et al. (12Balzer D. Ziegelin G. Pansegrau W. Kruft V. Lanka E. Nucleic Acids Res. 1992; 20: 1851-1858Crossref PubMed Scopus (125) Google Scholar). DNA bands were visualized by staining with ethidium bromide. The crystal structure of KorB-C, the C-terminal domain of the RP4-encoded repressor protein KorB, was determined in two space groups. After refinement, all residues lay in the allowed or favored region in the Ramachandran plot for both structures. Stereochemical parameters were calculated by PROCHECK (27Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and WHATCHECK (28Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 391: 272Crossref Scopus (1818) Google Scholar) and were in the range expected for structures with similar resolution. The orthorhombic structure was solved by multiple isomorphic replacement with contribution from the anomalous scattering of the platinum atoms. The asymmetric unit contains four copies of KorB-C. The final model consists of 1803 atoms and 250 solvent atoms. Of the expected 62 residues (297–358 of KorB), not all are visible in the electron density for each molecule. The N-terminal polypeptide region varies most, with molecule A starting from residue 305, molecules B and C from 302, and molecule D from 300. A number of side chains are not visible in the N-terminal region as well: the side chains of Lys305 and Lys306 of molecule A; those of Asp302, Lys303, and Lys306 of molecule B; those of Asp302, Lys303, and Lys305 of molecule C; and finally those of Asp300, Lys303, Lys305, Lys306, and Glu315 of molecule D. The flexibility of this part of the structure is also reflected in highB values in this area. The atomic displacement parameters assume values close to the mean B for all protein atoms at the start of strand β1. Increased B values are also observed in the loops and turns and near the C terminus. At a resolution of 1.7 Å, the refinement converged with a finalR value of 19.3% and an Rfree of 22.8% (Table II). The diffraction component precision index (29Cruickshank D.W.J. Acta Crystallogr. D. 1999; 55: 583-601Crossref PubMed Scopus (501) Google Scholar) was 0.17 Å.Table IIRefinement statisticsData and modelP212121P65Resolution range (Å)22–1.724–2.2No. of reflections32,1607,157R (work)/ R (free) [%]19.28/22.8015.17/22.70Side chains with alternative conformations116Protein atoms1833908Water molecules250120B values (Å2) Main chain atoms A/B/C/D22.96/24.09/21.91/25.3635.61/34.94 All23.6035.27 Side chain atoms A/B/C/D27.81/27.80/28.83/30.7542.73/40.99 All28.7541.88 All protein atoms A/B/C/D25.46/25.98/25.36/28.0239.28/37.97 All26.2238.63 All other atoms35.3244.33 All atoms27.3139.26r.m.s. deviations from targets Bond lengths (Å)0.0110.013 Bond angles (degrees)2.0982.528 Open table in a new tab The hexagonal structure of KorB-C was solved by molecular replacement based on the orthorhombic structure. The two KorB-C chains in the final model start with residues Asp300 and Pro299, respectively, in molecules A and B. As in the orthorhombic structure, some side chains in the N-terminal region are not visible, namely those of Lys305 in molecule A and those of Pro299, Lys305, and Lys306 in molecule B. The final model contains 968 protein and 120 solvent atoms. In P65, this structure was determined at 2.15-Å resolution with finalR/Rfree values of 15.2/22.7% and a diffraction component precision index of 0.28 Å. The following presentation of the KorB-C structure refers to chain D of the orthorhombic crystal form and the associated B/D dimer unless otherwise indicated, since they represent the most complete models seen at the higher resolution. KorB-C is folded into a five-stranded antiparallel all-β structure (Fig. 1). Strands β1, β2, β3, and β4 are arranged as an antiparallel up-and-down β-sheet with intervening loops of different length. The curvature of the strands and the pronounced left-handed twist of the sheet permit strand β5 to complete the fold by an antiparallel interaction on the free side of β1. Strands β1 and β2 are connected by loop L1 comprised of a β-turn (His313–Arg316), whereas a 10-residue loop (L2) links β2 to β3. This loop contains two β-turns, from Ile321 to Arg324 and from Ala328 to Tyr331. The short loop L3 is stabilized by a network of hydrogen bonds linking Tyr336oxygen with Gly340 nitrogen, Asp338Oδ1 with Asp339 nitrogen, and Asp339 Oδ2 with Gln341 nitrogen. Finally, the connection L4 between strands β4 and β5 contains a 310 helix of three residues (Leu347–Asp349). The KorB-C monomer is observed in six crystallographically independent copies, four times in the orthorhombic structure and twice in the hexagonal structure. All six copies share the general structural organization described above. This is evident from the root mean square (r.m.s.) deviations between equivalent atoms in the six molecules after least-squares superposition (Table III). α-Carbon positions superimpose with r.m.s. distances of 0.5 ± 0.2 Å and all equivalent atoms with 0.95 ± 0.2 Å for all chains with the exception of chain A of orthorhombic KorB-C. This chain clearly differs in the conformation of its three C-terminal residues, but it is structurally similar to the other five elsewhere. The unique conformation of the C-terminal tripeptide of molecule A is not caused by unusual crystal contacts. Instead, it seems that the disorder of this molecule's N-terminal part, for which electron density is observed only from residue Lys305 onward, is correlated to the altered C-terminal structure of molecule A of KorB-C. Otherwise, the most pronounced differences between KorB-C monomers are in the N-terminal peptide region, which, in the orthorhombic structure, adopts an extended conformation for chains C and D (see Fig. 3a), whereas it is less ordered in chains A and B. The extended N-terminal peptide of monomers C and D is stabilized by interactions with loop L2 from the other subunit of a dimer (see below) and interacts with the C-terminal strand β5.Table IIIStructural comparison of KorB-C monomersP65P212121ABABCDP65A0.7901.4360.9751.1250.971B0.3011.5321.1721.1081.015P212121A1.1881.2101.4881.6231.437B0.5550.6421.2261.0110.911C0.5470.6601.3080.4110.994D0.5360.5621.2600.3650.326The least-squares superposition used residues 306–358 for each monomer. Values are r.m.s. deviation (Å). Values below and above the diagonal are for Cα and all atoms, respectively. Open table in a new tab The least-squares superposition used residues 306–358 for each monomer. Values are r.m.s. deviation (Å). Values below and above the diagonal are for Cα and all atoms, respectively. The DALI server (30Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1291) Google Scholar) was used to identify structural similarities between KorB-C and other proteins. This search clearly indicated significant similarity of the KorB-C structure with the SH3 fold, predominantly known from protein domains involved in eukaryotic signal transduction (31Musacchio A. Wilmanns M. Saraste M. Prog. Biophys. Mol. Biol. 1994; 61: 283-297Crossref PubMed Scopus (144) Google Scholar) but occasionally also encountered in proteins from prokaryotes (32Baumann H. Knapp S. Lundbäck T. Ladenstein R. Härd T. Nat. Struct. Biol. 1994; 1: 808-819Crossref PubMed Scopus (157) Google Scholar, 33Falzone C.J. Kao Y.-H. Zhao J. Bryant D.A. Lecomte J.T.J. Biochemistry. 1994; 33: 6052-6062Crossref PubMed Scopus (72) Google Scholar, 34Safro M. Mosyak L. Protein Sci. 1995; 4: 2429-2432Crossref PubMed Scopus (19) Google Scholar, 35Whisstock J.C. Lesk A.M. Trends Biochem. Sci. 1999; 24: 132-133Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Using chain D of the orthorhombic KorB-C structure, the four top hits are characterized by DALI Z scores of 4.8 for a mutant human tyrosine kinase (PDB entry 1LCK; Ref. 36Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (240) Google Scholar), 4.5 for E. colidihydrofolate reductase (1VIE; Ref. 37Narayana N. Matthews D.A. Howell E.E. Nguyen-huu X. Nature Struct. Biol. 1995; 2: 1018-1025Crossref PubMed Scopus (76) Google Scholar), 4.4 for HIV-1 integrase (1IHV; Ref. 38Lodi P.J. Ernst J.A. Kuszewski J. Hickman A.B. Engelman A. Craigie R. Clore G.M. Gronenborn A.M. Biochemistry. 1995; 34: 9826-9833Crossref PubMed Scopus (271) Google Scholar), and 4.4 for the N-terminal SH3 domain of the proto-oncogene product c-Crk (1CKA; Ref. 39Wu X. Knudsen B. Feller S.M. Zheng J. äali A. Cowburn D. Hanafusa H. Kuriyan J. Structure. 1995; 3: 215-226Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Least-squares superpositions of KorB-C with these domains yield r.m.s. deviations between corresponding Cα atoms of 2.4 Å for the tyrosine kinase domain, 1.8 Å for the dihydrofolate reductase domain, 2.0 Å for HIV-1 integrase, and 1.8 Å for the SH3 domain from c-Crk. All of these structures are characterized by the typical antiparallel up-and-down β-sheet arrangement, which, in some cases, appears split in two sheets (one three-stranded and the other two-stranded) crossing each other at right angles. In addition, a three-residue 310 helix is often present between strands β4 and β5. These features are perfectly conserved in the crystal structure of KorB-C (Fig. 2). Missing in the KorB-C structure is the long loop between strands β1 and β2 that is known to be responsible for the binding of eukaryotic SH3 domains to the proline-rich motifs of other proteins during signal transduction (31Musacchio A. Wilmanns M. Saraste M. Prog. Biophys. Mol. Biol. 1994; 61: 283-297Crossref PubMed Scopus (144) Google Scholar). A second difference is the elongated strand β5 of KorB-C. This strand is crucial for the dimerization of KorB-C (see below). In addition, there is an N-terminal elongation of the polypeptide chain of KorB-C, which will make the connection to the preceding KorB domain and is very flexible in the isolated C-terminal domain. In the structure of KorB-C determined from orthorhombic crystals, the molecules in the asymmetric unit are related by noncrystallographic dyad axes. The KorB-C molecules in pairs A/C and B/D each are related by a 2-fold axis. A third 2-fold axis is found between the molecules A and C and, respectively, B and D. This axis is nearly, but not perfectly, parallel to the other axes and does not, therefore, establish 222-point symmetry. Instead, there are two similar pairs of KorB-C molecules, A/C and B/D (Fig.3a). In the hexagonal crystal form, two KorB-C molecules comprise the asymmetric unit. These two molecules (A and B) share the same arrangement with respect to each other with the molecule pairs A/C and B/D from the orthorhombic structure. The arrangement of six independent KorB-C molecules in two different crystal forms (P212121 and P65) thus follows one common pattern: a dimer formation involving molecule pairs A/C and B/D of orthorhombic KorB-C and A/B of hexagonal KorB-C, which is promoted mainly by interactions of residues from the C-terminal strand, β5. To examine which intermolecular interactions stabilize the observed dimer-like arrangement of KorB-C molecules (Fig.3b), the subunit interface was analyzed by the approach of Jones and Thornton (40Jones S. Thornton J.M. Prog. Biophys. Mol. Biol. 1995; 63: 31-165Crossref PubMed Scopus (498) Google Scholar, 41Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2289) Google Scholar) using their Protein-Protein Interactions server (available on the Internet at www. biochem.ucl.ac.uk/bsm/PP/). According to this analysis, 990 ± 90 Å2 of accessible surface are buried in the subunit interface for each of the six independent KorB-C subunits, corresponding to 23 ± 1% of the total accessible surface. The subunit interface area has a length of 31 ± 3 Å and a breadth of 25 ± 1 Å, and 63 ± 5% of the interface is contributed b" @default.
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• W2010707114 title "An Src Homology 3-like Domain Is Responsible for Dimerization of the Repressor Protein KorB Encoded by the Promiscuous IncP Plasmid RP4" @default.
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• W2010707114 doi "https://doi.org/10.1074/jbc.m110103200" @default.
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