NMR Structure of the R-module
2006; Elsevier BV; Volume: 281; Issue: 11 Linguagem: Inglês
10.1074/jbc.m510069200
ISSN1083-351X
AutoresFinn L. Aachmann, Britt I.G. Svanem, Peter Güntert, Steffen B. Petersen, Svein Valla, Reinhard Wimmer,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoIn the bacterium Azotobacter vinelandii, a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7) has been identified. These epimerases are responsible for the epimerization of β-d-mannuronic acid to α-l-guluronic acid in alginate polymers. The epimerases consist of two types of structural modules, designated A (one or two copies) and R (one to seven copies). The structure of the catalytically active A-module from the smallest epimerase AlgE4 (consisting of AR) has been solved recently. This paper describes the NMR structure of the R-module from AlgE4 and its titration with a substrate analogue and paramagnetic thulium ions. The R-module folds into a right-handed parallel β-roll. The overall shape of the R-module is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicated possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule and the importance of calcium binding for the stability of the R-module. Structure calculations showed that calcium ions can be incorporated in these loops without structural violations and changes. Based on the structure and the electrostatic surface potential of both the A- and R-module from AlgE4, a model for the appearance of the whole protein is proposed. In the bacterium Azotobacter vinelandii, a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7) has been identified. These epimerases are responsible for the epimerization of β-d-mannuronic acid to α-l-guluronic acid in alginate polymers. The epimerases consist of two types of structural modules, designated A (one or two copies) and R (one to seven copies). The structure of the catalytically active A-module from the smallest epimerase AlgE4 (consisting of AR) has been solved recently. This paper describes the NMR structure of the R-module from AlgE4 and its titration with a substrate analogue and paramagnetic thulium ions. The R-module folds into a right-handed parallel β-roll. The overall shape of the R-module is an elongated molecule with a positively charged patch that interacts with the substrate. Titration of the R-module with thulium indicated possible calcium binding sites in the loops formed by the nonarepeat sequences in the N-terminal part of the molecule and the importance of calcium binding for the stability of the R-module. Structure calculations showed that calcium ions can be incorporated in these loops without structural violations and changes. Based on the structure and the electrostatic surface potential of both the A- and R-module from AlgE4, a model for the appearance of the whole protein is proposed. Alginates are unbranched copolymers of 1→4-linked β-d-mannuronic acid (M) 3The abbreviations used are: M, β-d-mannuronic acid; G, α-l-guluronic acid; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RTX, repeats in toxins.3The abbreviations used are: M, β-d-mannuronic acid; G, α-l-guluronic acid; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RTX, repeats in toxins. and its C-5 epimer α-l-guluronic acid (G) (1Fischer F.G. Dorfel H. Hoppe Seyler's Z. Physiol. Chem. 1955; 302: 186-203Crossref PubMed Scopus (57) Google Scholar, 2Hirst E.L. Percival E. Wold J.K. J. Chem. Soc. 1964; : 1493-1499Crossref Google Scholar). Alginates are initially produced as a linear polymer of M-subunits only. Thereafter, single M-subunits in the polymeric chain are converted to G-subunits by enzymes called mannuronan C-5 epimerases. The mechanical and chemical properties of alginates depend on the composition and sequence of the two subunits, in particular, whether they are arranged in MM-, MG-, or GG-blocks. GG-blocks are rigid and form gels with divalent cations such as calcium. Recently, MG-blocks were suggested also to form gels with calcium (3Donati I. Holtan S. Morch Y.A. Borgogna M. Dentini M. Skjåk-Bræk G. Biomacromolecules. 2005; 6: 1031-1040Crossref PubMed Scopus (273) Google Scholar). Poly(M) regions, however, are more flexible without the ability to form gels with cations (4Grant G.T. Morris E.R. Rees D.A. Smith P.J.C. Thorn D. FEBS Lett. 1973; 32: 195-198Crossref Scopus (2181) Google Scholar, 5Stokke B.T. Smidsrød O. Bruheim P. Skjåk-Bræk G. Macromolecules. 1991; 24: 6637-6645Google Scholar). The relative amounts and distribution of M and G vary extensively among different species of both brown algae (6Haug A. Larsen B. Smidsrød O. Carbohydr. Res. 1974; 32: 217-225Crossref Scopus (387) Google Scholar, 7Hellebust J.A. Haug A. Can. J. Bot. 1972; 50: 177-184Crossref Google Scholar, 8Smidsrød O. Draget K.I. Carbohydr. Eur. 1996; 14: 6-13Google Scholar) and bacteria (9Skjåk-Bræk G. Grasdalen H. Larsen B. Carbohydr. Res. 1986; 154: 239-250Crossref PubMed Scopus (235) Google Scholar, 10Sherbrock-Cox V. Russell N.J. Gacesa P. Carbohydr. Res. 1984; 135: 147-154Crossref PubMed Scopus (71) Google Scholar)Alginate is for brown algae essentially the same as cellulose is for trees and plants. The stripes of the algae are mainly G-rich, giving them the stiffness to work as a skeleton, whereas leaves are M-rich showing flexibility (11Frei E. Preston R.D. Nature. 1962; 196: 130-134Crossref Scopus (20) Google Scholar).In Azotobacter sp., alginate is used as a capsular polysaccharide (12Gorin P.A.J. Spencer J.F.T. Can. J. Chem. 1966; 44: 993-998Crossref Google Scholar, 13Cote G.L. Krull L.H. Carbohydr. Res. 1988; 181: 143-152Crossref Scopus (56) Google Scholar), likewise in Pseudomonas sp. (14Linker A. Jones R.S. Nature. 1964; 204: 187-188Crossref PubMed Scopus (103) Google Scholar, 15Govan J.R. Fyfe J.A. Jarman T.R. J. Gen. Microbiol. 1981; 125: 217-220PubMed Google Scholar, 16Fett W.F. Osman S.F. Fishman M.L. Siebles T.S. Appl. Environ. Microbiol. 1986; 52: 466-473Crossref PubMed Google Scholar). In Azotobacter vinelandii, the composition and function of the alginates vary significantly depending on the environmental conditions.Vegetatively growing cells produce alginates that form a loose capsule structure that is easily released into the growth medium. These alginates are typically M-rich (17Sadoff H.L. Bacteriol. Rev. 1975; 39: 516-539Crossref PubMed Google Scholar), but under certain conditions of environmental stress, the cells enter a resting stage designated the "cyst" stage. The cysts are surrounded by a rigid alginate-containing protective coat, and due to the expression of a family of mannuronan C-5 epimerases (the AlgE family, see next paragraph), this coat also contains alginates with GG-blocks and strong gel-forming properties (17Sadoff H.L. Bacteriol. Rev. 1975; 39: 516-539Crossref PubMed Google Scholar, 18Høidal H.K. Svanem B.I.G. Gimmestad M. Valla S. Environ. Microbiol. 2000; 2: 27-38Crossref PubMed Scopus (33) Google Scholar).In all known alginate-producing organisms, the alginates are modified at the polymer level by mannuronan C-5 epimerases converting M- to G-residues (19Valla S. Li J. Ertesvåg H. Barbeyron T. Lindahl U. Biochimie (Paris). 2001; 83: 819-830Crossref PubMed Scopus (41) Google Scholar). The periplasmic epimerase AlgG is a part of alginate biosynthesis and is conserved in all known alginate-producing bacteria (20Rehm B.H. Ertesvåg H. Valla S. J. Bacteriol. 1996; 178: 5884-5889Crossref PubMed Google Scholar). Recently, the AlgG epimerase structure from Pseudomonas aeruginosa was suggested to contain a right-handed β-helix (21Douthit S.A. Dlakic M. Ohman D.E. Franklin M.J. J. Bacteriol. 2005; 187: 4573-4583Crossref PubMed Scopus (24) Google Scholar). This protein has, in addition, been shown to be essential for the protection of the alginate polymer against degradation by an alginate lyase, AlgL (22Gimmestad M. Sletta H. Ertesvåg H. Bakkevig K. Jain S. Suh S.J. Skjåk-Bræk G. Ellingsen T.E. Ohman D.E. Valla S. J. Bacteriol. 2003; 185: 3515-3523Crossref PubMed Scopus (115) Google Scholar). Additionally, the bacterium A. vinelandii encodes a family of seven secreted and calcium ion-dependent mannuronan C-5 epimerases (AlgE1-7) (23Ertesvåg H. Høidal H.K. Schjerven H. Svanem B.I.G. Valla S. Metab. Eng. 1999; 1: 262-269Crossref PubMed Scopus (52) Google Scholar). These epimerases consist of two types of structural modules, designated "A" (∼385 amino acids each, with one or two copies) and "R" (∼155 amino acids each, with one to seven copies), and additionally, a C-terminal signal peptide in the last of the R-modules (24Ertesvåg H. Doseth B. Larsen B. Skjåk-Bræk G. Valla S. J. Bacteriol. 1994; 176: 2846-2853Crossref PubMed Google Scholar, 25Ertesvåg H. Høidal H.K. Hals I.K. Rian A. Doseth B. Valla S. Mol. Microbiol. 1995; 16: 719-731Crossref PubMed Scopus (114) Google Scholar). The A-modules alone are catalytically active, but their reaction rates are significantly increased when covalently bound to at least one R-module (26Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). The R-module of AlgE4 is not catalytically active on its own (26Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). The N-terminal region of each R-module consists of four to seven copies of a nonapeptide with the consensus sequence LXG-GAGXDX, which is involved in calcium binding (24Ertesvåg H. Doseth B. Larsen B. Skjåk-Bræk G. Valla S. J. Bacteriol. 1994; 176: 2846-2853Crossref PubMed Google Scholar, 26Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). The individual sequence, number, and distribution of A- and R-modules give the individual epimerases their characteristic modes of action on this substrate. The R-module appears to have a significant role in the reaction catalyzed by the A-module by reducing the Ca2+ concentration needed for full activity and by enhancing the reaction rate (26Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). Recent experiments have also shown that the R-module influences the product specificity of the attached A-module, i.e. the degree of GG- or MG-blocks produced (27Bjerkan T.M. Bender C.L. Ertesvåg H. Drabløs F. Fakhr M.K. Preston L.A. Skjåk-Bræk G. Valla S. J. Biol. Chem. 2004; 279: 28920-28929Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).Therefore, it is essential to study the structure of such an alginate epimerase and its modules in detail to achieve insight into its function. Knowledge of the three-dimensional structure of these proteins opens a range of further investigations concerning the metal binding properties and the substrate binding of alginate epimerases. Of special interest is the role of the R-module(s) after secretion and its/their influence on the function of the A-module(s).Unsuccessful attempts have been made to crystallize the smallest epimerase (AlgE4 consisting of one A- and one R-module). However, the A-module has been produced and crystallized separately, and recently the structure of this module was solved by x-ray crystallography (28Bjerkan T.M. Structure-Function Analyses of Mannuronan C-5 Epimerases. Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway2003Google Scholar). The A-module belongs to the family of pectate lyase folds. It folds as a single-stranded, right-handed parallel β-helix. This β-helix consists of 12 complete turns, made up of four β-strands each, except for the last turn at the C terminus, where the helix narrows down to three β-strands per turn. An N-terminal amphiphatic α-helix forms a cap to this part of the protein (28Bjerkan T.M. Structure-Function Analyses of Mannuronan C-5 Epimerases. Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway2003Google Scholar). Here, we report the structure of the R-module containing the C-terminal signal sequence and its possible calcium and alginate binding sites.MATERIALS AND METHODSSample Preparation—Cloning, production, and purification of the uniformly 13C/15N-labeled R-module (167 amino acids) and the sample conditions for the NMR measurements were described previously (29Aachmann F.L. Svanem B.I.G. Valla S. Petersen S.B. Wimmer R. J. Biomol. NMR. 2005; 31: 259Crossref PubMed Scopus (6) Google Scholar). Samples for NMR studies contained 1.6 mm R-module in 20 mm HEPES buffer with 50 mm CaCl2 at pH 6.9 in 95% H2O/5% D2O or 99.9% D2O. All NMR experiments were recorded at 298 K on a Bruker DRX600 spectrometer equipped with a 5-mm xyz-gradient TXI(H/C/N) probe.NMR Spectroscopy—The sequence-specific resonance assignments and the triple resonance NMR experiments used to obtain them are described in Ref. 29Aachmann F.L. Svanem B.I.G. Valla S. Petersen S.B. Wimmer R. J. Biomol. NMR. 2005; 31: 259Crossref PubMed Scopus (6) Google Scholar. Three-dimensional 13C- and 15N-edited 1H,1H-NOESY spectra were recorded in D2O and H2O, respectively. The 15N-1H heteronuclear NOEs were calculated from two independently measured and integrated 15N-HSQC spectra as the ratio of the peak volumes with and without 1H saturation. Nuclear magnetic relaxation (T1 and T2) measurements of 15N were obtained by exponential fitting of the peak intensities in 15N-HSQC spectra acquired with different relaxation delays (30Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1781) Google Scholar, 31Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (1999) Google Scholar).Metal Binding—The displacement of calcium ions in the R-module by paramagnetic thulium was achieved by adding small amounts of the aliquots of a 4.7 mm TmCl3 solution for the first three steps until an R-module:calcium:thulium molar ratio of 1:25:0.5 was reached. Thereafter, aliquots of 47 mm TmCl3 were added for the next thirteen steps until a (R-module:calcium:thulium) molar ratio of 1:25:10.5 was reached. Finally, aliquots of 470 mm TmCl3 solution were added in three steps to a final ratio of (R-module:calcium:thulium) 1:25:30.75. In all titration steps, thulium was added directly to the uniformly 15N-labeled R-module solution in the NMR sample tube in small volumes so that the increase in sample volume could be neglected. A 15N-HSQC spectrum was recorded for each of the 19 titration steps.Chemical shift changes in two-dimensional HSQC spectra were quantified by calculation of an absolute change in chemical shift as,ΔHSQC=ΔνH2+ΔνN2(Eq. 1) where ΔνH is the chemical shift change of HN atoms expressed in Hz, and ΔνH is the chemical shift change of N atoms expressed in Hz.Alginate Binding—The interaction between the R-module and alginate was investigated by adding small amounts of a 10 mm solution of pentameric β-d-mannuronic acid (M5), dissolved in a buffer identical to the NMR sample, to a solution of 0.15 mm U-15N-labeled R-module in 50 mm CaCl2, 20 mm HEPES, pH 6.9. M5 was added in six portions until an R-module:alginate molar ratio of 1:1.2 was reached. In all titration steps, alginate was added in small volumes so that the increase in sample volume could be neglected. A 15N-HSQC spectrum was recorded for each titration step, and spectral changes were quantified according to Equation 1.Structure Calculation—NOESY cross-peaks were identified, integrated, and assigned in the aforementioned NOESY spectra using the program XEASY (32Bartels C. Xia T.-H. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Crossref PubMed Scopus (1593) Google Scholar). The CALIBA (33Güntert P. Qian Y.Q. Otting G. Müller M. Gehring W. Wüthrich K. J. Mol. Biol. 1991; 217: 531-540Crossref PubMed Scopus (131) Google Scholar) subroutine in CYANA was used to convert cross-peak intensities from NOESY spectra into distance constraints. Backbone torsion angle restraints were obtained from secondary chemical shifts using the program TALOS (34Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2729) Google Scholar). On the basis of this input, the structure was calculated using the torsion angle dynamics program CYANA (35Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar). Weak restraints on (ϕ,Ψ) torsion angle pairs and on side chain torsion angles between tetrahedral carbon atoms were applied temporarily during the high temperature and cooling phases of the simulated annealing schedule to favor allowed regions of the Ramachandran plot and staggered rotamer positions, respectively. Structure calculations were started from 100 conformers with random torsion angle values. The 20 conformers with the lowest final CYANA target function values were embedded in a water shell of 8 Å thickness and energy-minimized against the AMBER force field (36Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11449) Google Scholar) with the program OPALp (37Koradi R. Billeter M. Güntert P. Comput. Phys. Commun. 2000; 124: 139-147Crossref Scopus (148) Google Scholar).Incorporation of Calcium Ions into the Structure Calculation—Additional structure calculations were performed with calcium ions incorporated into the structure. The calcium binding loops were determined from the thulium titration experiments. The binding geometry was inferred from alkaline metalloproteases from P. aeruginosa (Protein Data Bank (PDB) code 1KAP) and Serratia marcescens (PDB code 1SAT) (38Baumann U. J. Mol. Biol. 1994; 242: 244-251Crossref PubMed Scopus (159) Google Scholar, 39Baumann U. Wu S. Flaherty K.M. Mckay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (420) Google Scholar), two proteins with highly similar calcium binding motifs.The calcium ion van der Waals radius was set to 1.12 Å (40Shannon R.D. Acta Crystallogr. Sect. A. 1976; 32: 751-767Crossref Scopus (52815) Google Scholar). The positions of the Ca2+ ions were restrained by upper limit distance constraints between the respective Ca2+ ions and protein atoms using the calcium binding β-roll motifs from P. aeruginosa and S. marcescens (38Baumann U. J. Mol. Biol. 1994; 242: 244-251Crossref PubMed Scopus (159) Google Scholar, 39Baumann U. Wu S. Flaherty K.M. Mckay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (420) Google Scholar) as templates. One-half of the octahedral coordination is made up by the backbone oxygen atoms of G2 (2.5 Å upper distance limit), G4 (2.5 Å), and the side chain oxygen Oδ1 of D6 (2.5 Å). The coordination sphere is completed by the backbone oxygen atoms from G1′ (2.5 Å) and X3′ (2.5 Å) and the side chain oxygen Oδ2 of D6′ (3.2 Å) of the neighboring GGXGXD loop. To define the two last calcium binding sites, only distance constraints to the first half of the coordination sphere and D6′ were incorporated, because it was found in the x-ray structure of P. aeruginosa that the rest of the coordination sphere was made up by water ligands (39Baumann U. Wu S. Flaherty K.M. Mckay D.B. EMBO J. 1993; 12: 3357-3364Crossref PubMed Scopus (420) Google Scholar). Our structure calculations including Ca2+ ions are not intended to serve as a direct determination of the structural details of calcium binding, but merely to demonstrate the compatibility of our data with the known calcium binding mode of the nonapeptide motif.RESULTSThe solution structure of the R-module was calculated on the basis of NOE upper distance limits and TALOS-derived (34Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2729) Google Scholar) backbone torsion angle restraints (Table 1). The coordinate and constraint files were deposited in the PDB data base (accession code 2AGM).TABLE 1Characterization of energy-minimized NMR structuresWithout calcium ionsWith calcium ionsInput data for the structure calculationTotal number of NOE distance constraints1,5761,576 Intraresidual575575 Short range418418 Medium range130130 Long range453453Number of upper distance limits for calcium ions34Torsion angle constraintsaObtained from secondary chemical shifts using the program TALOS (34)138138Structure statistics, 20 conformersbThe values given are the average and standard deviation over the 20 energy-minimized conformers with the lowest CYANA (version 2.1) target function values that represent the NMR solution structureCYANA target function value (Å2)cThe average target function values for the 20 best CYANA conformers before energy minimization3.47 ± 0.534.60 ± 1.21Maximal distance constraint violation (Å)0.13 ± 0.010.14 ± 0.01Maximal torsion angle constraint violation (°)3.88 ± 0.603.95 ± 1.37AMBER energies (kcal/mol)Total-4,537 ± 161-7,769 ± 201van der Waals-397 ± 20-149 ± 24Electrostatic-5,289 ± 167-8,745 ± 180PROCHECK Ramachandran plot analysisResidues in favored regions (%)69.172.8Residues in additionally allowed regions (%)27.724.7Residues in generously allowed regions (%)2.92.2Residues in disallowed regions (%)0.30.3Root mean square deviation to the average coordinates (Å)dAverage coordinates of the 20 energy-refined conformers after superposition for best fit of the N, Cα, and C′ atoms of the residues indicated in parenthesesN, Cα, C′ (5–145)1.131.19N, Cα, C′ (secondary structure)eResidues 6–8, 15–17, 24–26, 33–35, 42–44, 51–53, 61–63, 66–68, 71–73, 81–83, 100–105, 111–115, and 127–1290.660.67Heavy atoms (5–145)1.661.68Heavy atoms (secondary structure)eResidues 6–8, 15–17, 24–26, 33–35, 42–44, 51–53, 61–63, 66–68, 71–73, 81–83, 100–105, 111–115, and 127–1291.201.21a Obtained from secondary chemical shifts using the program TALOS (34Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2729) Google Scholar)b The values given are the average and standard deviation over the 20 energy-minimized conformers with the lowest CYANA (version 2.1) target function values that represent the NMR solution structurec The average target function values for the 20 best CYANA conformers before energy minimizationd Average coordinates of the 20 energy-refined conformers after superposition for best fit of the N, Cα, and C′ atoms of the residues indicated in parenthesese Residues 6–8, 15–17, 24–26, 33–35, 42–44, 51–53, 61–63, 66–68, 71–73, 81–83, 100–105, 111–115, and 127–129 Open table in a new tab Fig. 1 shows the sequence and the location of the secondary structure elements, whereas Fig. 2, A and C, show the three-dimensional structure. The structured N-terminal end of the R-module is a β-roll defined by three amino acids forming a short β-strand and six amino acids forming a loop to the next short β-strand followed by six amino acids forming the next loop, resulting in an 18-amino-acid β-roll unit. This is reflected by 19.5% of all long range NOEs being observed between atoms that are 18 residues apart in the sequence. This structure is repeated three times (β1 6-8, β2 15-17, β3 24-26, β4 33-35, β5 42-44, β6 51-53) making up three turns of the β-roll (Figs. 1 and 3). Thereafter, a loop-out forms an anti-parallel β-hairpin (β7 61-63, β8 66-68). The polypeptide chain folds back and makes a new turn elongating the β-roll (β9 71-73, β10 81-83). Thereafter, a longer loop bulges out, followed by a less well defined region (91-100). The structure ends with two anti-parallel β-strands (β11 100-104, β12 111-114). The last β-strand (β13 127-129) is situated in between the roll and the anti-parallel β-sheet (β11-β12), parallel to the first and anti-parallel to the latter. Finally, the polypeptide again forms a somewhat less well ordered loop structure from residue 130 onward. From amino acid residue 145 to the C terminus (residue 167), no ordered secondary structure exists. This is in agreement with heteronuclear NOE measurements (data not shown) pointing at a more mobile polypeptide chain.FIGURE 2The NMR structure of the AlgE4 R-module. A, ribbon drawing of the energy-refined R-module structure with the lowest residual CYANA target function, viewed from the front and side of the β-roll. The secondary structure elements are parallel strands β1 (6-8), β2 (15-17), β3 (24-26), β4 (33-35), β5 (42-44), β6 (51-53), β9 (71-73), β10 (81-83), and β13 (125-129), and the anti-parallel strands β7 (61-63), β8 (66-68), β11 (100-104), and β12 (111-114) all shown in cyan. B, structure of heavy atoms in the loop region of residues 27-44 for the 20 best energy-refined conformations of the R-module (thin lines) and residues 351-368 of P. aeruginosa (thick lines). C, bundle of the 20 energy-refined conformations of the R-module in the region of amino acid residues 5-145. Shown are the R-module without calcium ions (left) and the R-module with calcium ions incorporated in the loops (right). β-sheet regions are colored in cyan and calcium ions are depicted as orange spheres. The coordinate files of the calcium-free form were deposited in the PDB data base with accession code 2AGM. D, electrostatic surface potential of the AlgE4 A- (left) (28Bjerkan T.M. Structure-Function Analyses of Mannuronan C-5 Epimerases. Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway2003Google Scholar) and R-modules (right). Red and blue, respectively, denote regions of negative and positive potential on the protein surface. E, Shown is the interaction with Tm3+. Green patches denote amino acids, with ΔHSQC > 20 Hz at an R-module:calcium:thulium ratio of 1:25:3.75, and red patches denote amino acids, with ΔHSQC > 100 Hz at an R-module:calcium:thulium ratio of 1:25:25.75. F, interaction with pentameric M5 alginate. Blue surface patches indicate amino acids, with ΔHSQC > 60 Hz, cyan patches indicate amino acids, with 30 Hz < ΔHSQC < 60 Hz. Green surface patches indicate amino acids that change significantly upon titration with M5 but only where this change could not be quantified throughout the titration because of signal overlap. ΔHSQC values were calculated according to Equation 1. Structure models were prepared with the program MOLMOL (60Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6469) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Observed chemical shift changes upon titration of the R-module with thulium. Shown are ΔHSQC values (calculated according to Equation 1) at R-module:calcium:thulium ratios of 1:25:3.75 (top) and 1:25:25.75 (bottom) plotted as a function of the amino acid sequence. Amino acids for which shifts could not be observed after thulium titration are as follows: 2, 4, 5, 7, 9, 12, 13, 14, 17, 30, 31, 34, 35, 47, 48, 51, 55, 58, 59, 74, 76, 78, 79, 80, 86, 94, 120, 121, 134, 153, 154, 156, 157, 159, 160, and 167. Underlined amino acid numbers indicate residues in a turn or coil structure. Doubly underlined amino acid numbers indicate residues in one of the loops of the repeat region. Secondary structure elements in the polypeptide chain are plotted along the top of the graph.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Remarkably, only 27% of the amino acids of the R-module are in regular secondary structure elements (17% in parallel and 10% in anti-parallel β-strands), whereas 73% of the residues form a coil structure. This is corroborated by circular dichroism measurements (results not shown). Altogether, the protein forms an elongated structure along the axis of the β-roll with a small groove at the front side. The electrostatic surface potential of the R-module shows a positively charged patch formed by arginine and lysine side chains along the small groove at the front side of the β-roll. Aspartic acid and glutamic acid residues form negatively charged patches along the turns of the β-roll (Fig. 2D, right).To obtain further information about the intramolecular dynamics of the R-module, 15N-1H-NOEs as well as T1 and T2 of 15N were measured (data not shown). The 15N-1H-NOEs show increased flexibility for the N-terminal residues 1-7 and a highly mobile chain from residue 145 onward. The same tendency is reflected in the 15N T1 and T2 relaxation times. For the structured part of the R-module, the T1:T2 ratio is a direct measure of the correlation time of the overall rotational tumbling of the molecule. The average T1:T2 ratio for the protein was 12.1 ± 1.1, which, assuming a spherical particle, corresponds to an overall rotational correlation time τc = 10.7 ± 0.5 ns (30Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1781) Google Scholar).The R-module has been shown to bind calcium ions (26Ertesvåg H. Valla S. J. Bacteriol. 1999; 181: 3033-3038Crossref PubMed Google Scholar). To obtain further information about calcium binding sites, it was attempted to prepare a metal-free apo-form of the R-module by adding EDTA. However, this resulted in immediate precipitation, so that no sample of apo-R-module could be obtained. The importance of Ca2+ ions for the stability of the R-module is also underlined by the fact that NMR samples of the R-module at 5 mm CaCl2 showed visible signs of degradation in the NMR spectra after 48 h at room temperature, whereas the NMR samples at 50 mm CaCl2 were stable for months. Thus, because an apo-R-module was beyond reach, a titration was performed substituting Ca2+ ions with a paramagnetic ion. We chose
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