NMR Dynamic Studies Suggest that Allosteric Activation Regulates Ligand Binding in Chicken Liver Bile Acid-binding Protein
2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês
10.1074/jbc.m513003200
ISSN1083-351X
AutoresLaura Ragona, Maddalena Catalano, Marianna Luppi, Daniel O. Cicero, Tommaso Eliseo, Jefferson Foote, Federico Fogolari, Lucia Zetta, Henriette Molinari,
Tópico(s)Metabolism and Genetic Disorders
ResumoApo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via 15N relaxation and steady state heteronuclear 15N(1H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the β-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His98, located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His98 to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His98 protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids. Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via 15N relaxation and steady state heteronuclear 15N(1H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the β-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His98, located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His98 to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His98 protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids. Recent studies have shown that bile acids not only serve as the physiological detergents that facilitate absorption, transport, and distribution of lipid-soluble vitamins and dietary fats but also are the signaling molecules that activate nuclear receptors and regulate bile acid and cholesterol metabolism. In addition, bile acids induce the cytochrome P450 3A family of cytochrome P450 enzymes that detoxify bile acids, drugs, and xenobiotics in the liver and intestine, induce hepatocyte apoptosis, and activate the gene encoding a candidate bile acid transporter protein (1Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2244) Google Scholar). Given the important role of bile acids, the study of their transport at a molecular level is of special medical and pharmacological interest. In this line it is essential to gain insight into the three-dimensional structures and dynamic behavior of proteins, in their free and complexed forms, involved in bile acid recycling. Interestingly, bile acids have been suggested to be the putative ligands of a group of intracellular lipid-binding proteins (iLBPs) 2The abbreviations used are:iLBP,intracellularlipid-bindingprotein;ASBT,apicalsodium-dependent bile salt transporter; cl-BABP, chicken liver bile acid-binding protein; FABP, fatty acid-binding protein; BABP, bile acid-binding protein; ILBP, ileal lipidbinding protein; MD, molecular dynamics; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; r.m.s.d., root mean square deviation; PDB, Protein Data Bank; MALDI, matrix-assisted laser desorption ionization; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence; BSEP, bile salt export pump. or fatty acidbinding proteins (FABP), expressed in the liver of nonmammalian species, and referred to previously as liver basic FABP. FABPs have been classified and described on the basis of the organ that they were initially isolated from, but several instances are known in which more than one FABP type has been shown to be produced by a single tissue. We have reported previously on the higher similarity of liver basic FABPs from nonmammalian species with ileal lipid-binding protein (ILBP) rather than with mammalian liver FABP (2Vasile F. Ragona L. Catalano M. Zetta L. Perduca M. Monaco H. Molinari H. J. Biomol. NMR. 2003; 25: 157-160Crossref PubMed Scopus (15) Google Scholar). In agreement with this observation, bile acid binding and transport is emerging as the specific function of the liver nonmammalian subfamily, hence called liver bile acid-binding protein (BABP) (2Vasile F. Ragona L. Catalano M. Zetta L. Perduca M. Monaco H. Molinari H. J. Biomol. NMR. 2003; 25: 157-160Crossref PubMed Scopus (15) Google Scholar, 3Nichesola D. Perduca M. Capaldi S. Carrizo M.E. Righetti P.G. Monaco H.L. Biochemistry. 2004; 43: 14072-14079Crossref PubMed Scopus (54) Google Scholar). At variance, the paralogous proteins expressed in the same tissue but in mammals play a role in fatty acid binding and transport (4Thompson J. Reese-Wagoner A. Banaszak L. Biochim. Biophys. Acta. 1999; 1441: 117-130Crossref PubMed Scopus (51) Google Scholar). A multiple alignment of all the known sequences of nonmammalian liver BABPs with ILBPs is reported in Fig. 1. It has been proposed that internal protein dynamics in iLBPs could be intimately connected with ligand recognition and interaction (2Vasile F. Ragona L. Catalano M. Zetta L. Perduca M. Monaco H. Molinari H. J. Biomol. NMR. 2003; 25: 157-160Crossref PubMed Scopus (15) Google Scholar, 5Bakowies D. van Gunsteren W.F. J. Mol. Biol. 2002; 315: 713-736Crossref PubMed Scopus (61) Google Scholar, 6Krishnan V.V. Sukumar M. Gierasch L.M. Cosman M. Biochemistry. 2000; 39: 9119-9129Crossref PubMed Scopus (41) Google Scholar, 7Tochtrop G.P. Bruns J.L. Tang C. Covey D.F. Cistola D.P. Biochemistry. 2003; 42: 11561-11567Crossref PubMed Scopus (38) Google Scholar, 8Tochtrop G.P. DeKoster G.T. Covey D.F. Cistola D.P. J. Am. Chem. Soc. 2004; 126: 11024-11029Crossref PubMed Scopus (53) Google Scholar). We report here a structural and dynamic study on chicken liver BABP (cl-BABP), in its apo- and holo-form, combining heteronuclear NMR experiments and 15N NMR relaxation measurements with MD simulations. We investigate the role of the protonation state of a buried histidine on protein dynamics. We discuss here the observed change in dynamics upon ligand binding in terms of an allosteric activation mechanism, i.e. a shift between inactive and active conformations (9Volkman B.F. Lipson D. Wemmer D.E. Kern D. Science. 2001; 291: 2429-2433Crossref PubMed Scopus (539) Google Scholar). The proposed mechanism for ligand binding in cl-BABP is further analyzed in light of data reported for other members of the iLBP family and discussed as functional to bile acid enterohepatic circulation. Protein Expression and Purification—Recombinant cl-BABP was expressed as soluble protein in Escherichia coli BL21 (DE3) bearing the recombinant plasmid pET24d. Transformed cells were grown on plates containing 50 μg/ml kanamycin. One liter of LB was inoculated with an overnight culture and incubated at 310 K until cells reached an A600 of 0.8. Protein expression was induced by addition of 0.7 mm isopropylthiogalactopyranoside and incubation continued overnight at 293 K. The cells were harvested and resuspended in lysis buffer (50 mm Tris, 10% sucrose, 1 mm EDTA, 10 mm β-mercaptoethanol, pH 8.0). After lysis, the supernatant, containing cl-BABP, was loaded on a DEAE-cellulose (Whatman) anion exchange column equilibrated with 50 mm Tris acetate, pH 7.8. The same buffer was used for protein elution. Fractions containing cl-BABP were concentrated and resolved on a Sephacryl S-100 HR (Amersham Biosciences) column equilibrated with 50 mm Tris-HCl, 0.2 m NaCl, pH 7.2. cl-BABP was delipidated as described (10Glatz J.F. van der Vusse G.J. Prog. Lipid Res. 1996; 35: 243-282Crossref PubMed Scopus (490) Google Scholar). The protein purity was checked by the presence of a single band on SDS-PAGE and by mass spectrometry. The protein yields were 90 mg/liter of bacterial culture. 15N isotope labeling was achieved using M9 minimal media containing 1 g/liter 15NH4Cl, following protocols reported in the literature (11Marley J. Lu M. Bracken C. J. Biomol. NMR. 2001; 20: 71-75Crossref PubMed Scopus (630) Google Scholar). The extent of 15N labeling was verified by MALDI mass analysis, and the isotope incorporation was found to be more than 92%. [15N]cl-BABP was obtained in a yield of 50 mg/liter of minimal media. 13C,15N double labeling was obtained with the same procedure using M9 minimal media containing 1 g/liter 15NH4Cl and 4 g/liter 13C-enriched sucrose. The extent of labeling, verified by MALDI mass analysis, was >90%, and yields of 25 mg/liter of minimal media were obtained. Commercial chenodeoxycholic acid (Sigma) was employed for the preparation of holo-cl-BABP with a ligand to protein ratio 5:1, as described previously (8Tochtrop G.P. DeKoster G.T. Covey D.F. Cistola D.P. J. Am. Chem. Soc. 2004; 126: 11024-11029Crossref PubMed Scopus (53) Google Scholar). NMR Experiments—NMR data were recorded on Bruker Avance 500, 600, and 700 MHz spectrometers equipped with pulse field gradient triple-resonance probes. 0.5 mm protein samples in phosphate buffer at pH 7.0 and 5.6 and 298 K were employed for structure determination and relaxation measurements. Two-dimensional homonuclear TOCSY (mixing 70 ms) and NOESY (mixing 150 ms) were performed at 500 and 700 MHz on cl-BABP sample at pH 7.0, 298 K. Water suppression was achieved using the excitation sculpting sequence (12Prost E. Sizun P. Piotto M. Nuzillard J.M. J. Magn. Reson. 2002; 159: 76-81Crossref PubMed Scopus (26) Google Scholar) for TOCSY and WATERGATE (13Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3563) Google Scholar) for NOESY. Two-dimensional homonuclear TOCSY and NOESY were also performed at 298 K and pH 5.6, i.e. in the same conditions as those reported previously for the purified protein (2Vasile F. Ragona L. Catalano M. Zetta L. Perduca M. Monaco H. Molinari H. J. Biomol. NMR. 2003; 25: 157-160Crossref PubMed Scopus (15) Google Scholar). For the 15N-enriched apo-cl-BABP (pH 5.6 and pH 7) and holo-cl-BABP (pH 7.0) samples, 15N HSQC-TOCSY (14Marion D. Driscoll P.C. Kay L.E. Wingfield P.T. Bax A. Gronenborn A.M. Clore G.M. Biochemistry. 1989; 28: 6150-6156Crossref PubMed Scopus (944) Google Scholar) (mixing 85 ms), 1H-15N HSQC-NOESY (14Marion D. Driscoll P.C. Kay L.E. Wingfield P.T. Bax A. Gronenborn A.M. Clore G.M. Biochemistry. 1989; 28: 6150-6156Crossref PubMed Scopus (944) Google Scholar) (mixing 150 ms), and HANNAH values (15Kuboniwa H. Grzesiek S. Delaglio F. Bax A. J. Biomol. NMR. 1994; 4: 871-878Crossref PubMed Scopus (336) Google Scholar) were recorded. The following triple resonance experiments, using standard parameter sets (16Pelton J.G. Torchia D.A. Meadow N.D. Wong C.Y. Roseman S. Biochemistry. 1991; 30: 10043-10057Crossref PubMed Scopus (62) Google Scholar), were recorded on the doubly labeled [15N,13C]apo- and -holo-cl-BABP in H2O at 700 MHz and at pH 7.0: HNCA, HN(CO)CA, HNCO, CBCANH, and CBCA(CO)NH. For the sample dissolved in D2O HACACO, (H)CCH-COSY, (H)CCH-TOCSY, H(C)CH-COSY, and H(C)CH-TOCSY experiments were performed (17Powers R. Clore G.M. Bax A. Garrett D.S. Stahl S.J. Wingfield P.T. Gronenborn A.M. J. Mol. Biol. 1991; 221: 1081-1090Crossref PubMed Scopus (30) Google Scholar). Two NOESY-type three-dimensional experiments (mixing 100 ms) were acquired, one optimized for aliphatic and one for aromatic residues. A series of two-dimensional 1H-15N HSQC experiments was performed for the apoprotein at different pH values (in the range 4.2-7.4) to allow for measurement of the midpoint of the chemical shift pH-driven titration. Spectra were assigned on the basis of the assignments obtained at pH 7.0 and 5.6. The 15N chemical shift titration data were fitted to Equation 1 to evaluate pKa values (18Hass M.A. Thuesen M.H. Christensen H.E. Led J.J. J. Am. Chem. Soc. 2004; 126: 753-765Crossref PubMed Scopus (35) Google Scholar), δobs=δd+δp-δd1+10(pH-pKa)(Eq. 1) where δp and δd are the chemical shifts of the protonated and the deprotonated state, respectively. Calculation of 1H and 15N secondary shifts was performed according to d=((ΔdHN2+ΔdN2/25)/2)1/2 (19Cicero D.O. Melino S. Orsale M. Brancato G. Amadei A. Forlani F. Pagani S. Paci M. Int. J. Biol. Macromol. 2003; 33: 193-201Crossref PubMed Scopus (7) Google Scholar). 15N relaxation experiments (20Farrow 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 (2030) Google Scholar), run as water flip-back version, were acquired at 600 and 700 MHz both at pH 7.0 and 5.6. Eleven delays (2.5, 20, 60, 100, 150, 200, 300, 400, 600, 800, and 1000 ms) were used for T1 measurements, and nine delays (16.96, 33.92, 50.80, 67.84, 101.76, 135.68, 169.6, 220.48, and 237.44 ms) were used for T2 measurements. The delay in the Carr-Purcell-Meiboom-Gill pulse train was set to 0.45 ms. 1H-15N NOE experiments were acquired with an overall recycling delay of 6 s (20Farrow 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 (2030) Google Scholar). To analyze the exchange contribution to relaxation at pH 7, T1, T2, and 1H-15N NOEs were also measured at 500 MHz, in the same conditions as described for higher field measurements. Relaxation measurements were identically performed at pH 7.0 for holo-cl-BABP complexed with chenodeoxycholate. Data were processed with XWINNMR and NMRPipe (21Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11837) Google Scholar) and analyzed with NMR-View 5.0.3 software package (22Johnson B.A. Methods Mol. Biol. 2004; 278: 313-352PubMed Google Scholar). Structure Calculation of Apo-cl-BABP—Volume integration performed on was the three-dimensional 15N-13C NOESY and 1H-15N HSQC-NOESY spectra using NMRView (22Johnson B.A. Methods Mol. Biol. 2004; 278: 313-352PubMed Google Scholar). Peak volume calibration was performed using the median method and a routine of NMRView program, and the obtained list of distances was used as input for DYANA (23Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2569) Google Scholar) calculations. φ angle restraints were derived from JHN,HA coupling constants estimated from three-dimensional HNHA experiments (15Kuboniwa H. Grzesiek S. Delaglio F. Bax A. J. Biomol. NMR. 1994; 4: 871-878Crossref PubMed Scopus (336) Google Scholar). φ angle restraints of 139 ± 30 ° for Jhn,ha coupling constants greater than 8.0 Hz and 60 ± 30 ° for Jhn,ha coupling constants smaller than 5.0 Hz were used as restraints. Amide proton exchange rates were estimated from a series of 1H-15N HSQC spectra performed at different times after dissolving the protein in D2O (data not shown). The partners for all hydrogen bonds were assigned on the basis of preliminary structures obtained by imposing only NOE restraints. Each hydrogen bond was introduced as a restraint on O-N distance of 3.00 Å and HN-O distance of 2.00 Å. The decision was taken to introduce in the calculation only totally unambiguous restraints, i.e. those correlations that were not affected by overlap in any spectra. The restraints were re-examined to check for consistent violations. One hundred calculations were run employing DYANA (23Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2569) Google Scholar), and the 20 conformers with the lowest residual target function were analyzed. The 20 final DYANA structures were further refined using the AMBER force field, as implemented in the program DISCOVER (Molecular Simulations, San Diego). A dielectric constant of 4 × r was used, and a scaling factor of 10 was used for out-of-plane interactions. Each structure was minimized performing 100 steps of steepest descent and 300 steps of conjugate gradient. The 10 structures with the lowest potential energy were selected for further analysis. The structures were deposited in the PDB with code 1zry. Relaxation Data Analysis—Relaxation times were calculated via least squares fitting of peak intensities, using the rate analysis routine of NMRView program (22Johnson B.A. Methods Mol. Biol. 2004; 278: 313-352PubMed Google Scholar). The heteronuclear NOE effects were calculated from the ratio of cross-peak intensities in spectra collected with and without amide proton saturation. The principal components of cl-BABP inertia tensor were calculated using Pdbinertia (A. G. Palmer III, Columbia University). The principal moments of inertia of apo-cl-BABP at pH 7 were calculated on the basis of our NMR structure (PDB code 1zry), whereas at pH 5.6 the representative coordinates from MD simulations were used (see below). For holoprotein, the x-ray structure (PDB code 1tw4) was employed. Isotropic and anisotropic models were tested for apo- and holo-cl-BABP. An initial estimate of the overall correlation time and of principal components and orientation of the diffusion tensor can be reliably determined from the angular dependence of the relaxation rates of a subset of NH vectors assumed to have a negligible component of internal motion and/or exchange contribution to 15N relaxation. The selection of the subset of residues was made following the procedures described in the literature (24Pawley N.H. Wang C. Koide S. Nicholson L.K. J. Biomol. NMR. 2001; 20: 149-165Crossref PubMed Scopus (71) Google Scholar): residues with NOE c12I0/d12 will have an exchange contribution. However, taking in consideration experimental and fitting errors, a threshold of 1.3 × 〈m 〉 was used to determine residues subject to exchange (29Phan I.Q.H. Boyd J. Campbell I.D. J. Biomol. NMR. 1996; 8: 369-378Crossref PubMed Scopus (87) Google Scholar), where 〈m 〉 is the average slope. Theoretical pKa Calculations—All pKa calculations have been performed as described previously (30Antosiewicz J. McCammon J.A. Gilson M.K. J. Mol. Biol. 1994; 238: 415-436Crossref PubMed Scopus (764) Google Scholar, 31Fogolari F. Esposito G. Viglino P. Molinari H. J. Comput. Chem. 2001; 22: 1830-1842Crossref PubMed Scopus (20) Google Scholar). The linear Poisson-Boltzmann equation was solved for different charge states, and the electrostatic free energy was used to estimate pKa shifts. The mid-point of the titration for each site is taken as its pKa value. All Poisson-Boltzmann calculations have been performed using the program UHBD (32Wade R.C. Luty B.A. Demchuk E. Madura J.D. Davis M.E. Briggs J.M. McCammon J.A. Nat. Struct. Biol. 1994; 1: 65-69Crossref PubMed Scopus (66) Google Scholar). Molecular Dynamics Simulations—Molecular dynamics simulations were performed using the program GROMACS (version 3.2.1) employing the GROMACS force field (ffgmx2) (33van Aalten D.M. Findlay J.B. Amadei A. Berendsen H.J. Protein Eng. 1995; 8: 1129-1135Crossref PubMed Scopus (176) Google Scholar). The protocol used was essentially as described previously for β-lactoglobulin (34Fogolari F. Moroni E. Wojciechowski M. Baginski M. Ragona L. Molinari H. Proteins. 2005; 59: 91-103Crossref PubMed Scopus (28) Google Scholar). The structure of the bile acid-binding protein was taken from PDB code 1zry, model 1. Protons were added using the program pdb2gmx, in the GRO-MACS suite of programs, for optimization of the hydrogen bond network. The protein was first minimized by 200 steepest descent minimization steps, followed by 200 conjugate gradients steps. Because of lack of solvent in this step, the dielectric constant used was 10. The Poisson Boltzmann equation was used to compute the electrostatic potential around the molecule. The lowest potential region at 0.7 nm from any protein atom was chosen for placing a counterion. The procedure was repeated on the protein and ion(s) until the net charge of the system was 0. The minimized protein and ions were then solvated in a box of SPC water with boundaries at least 1.6 nm away from any protein or ion atom. After addition of solvent molecules and ions to the system, long range electrostatic interactions were treated by particle mesh Ewald method with the following parameters: distance for non-bond interaction cutoff 12 Å and spacing for the fast Fourier transform grid 1.2 Å. The solutes were fixed, and water was energy-minimized by 100 steepest descent minimization steps. A short molecular dynamics run (50 ps) keeping the solutes fixed was performed to let the water soak the system. During this run the time step was set to 1 fs. Finally, the unrestrained system was energy minimized by 200 steepest descent steps and equilibrated in the NTP ensemble for 100 ps. In all molecular dynamics simulations the system was in equilibrium with a temperature bath at 300 K, with relaxation time constant of 0.1 ps. The system compressibility was that of water, 4.5 × 10-5 bar-1. The relaxation time for pressure equilibration was 0.5 ps. The initial velocities were set to 0. Two 3.6-ns MD simulations were performed for the low pH form (with the two histidines protonated) and the neutral pH form (with both histidines deprotonated) of cl-BABP. In both cases 100 ps of equilibration time were employed. The r.m.s.d. from starting structure could be fitted by an exponential with a time constant of 150 ps for both simulated forms, although for the protonated form a much slower, very small but detectable, increase in r.m.s.d. was observed throughout the run. The backbone r.m.s.d. from native, including protein ends and loops, is fluctuating around 2.2 Å after few hundred ps. To make sure that the system was equilibrated (at least in this time range), we repeated all analyses of local fluctuations for the same trajectories truncated at 1.8 ns. No significant difference was found. Snapshots were taken at 100-ps intervals along the simulations, and these 37 snapshots were used for structural analysis. The snapshot exhibiting the smaller average r.m.s.d. with respect to all other snapshots has been taken as the most representative structure in the ensemble. All structural analysis have been performed using the program Mol-Mol (36Musafia B. Buchner V. Arad D. J. Mol. Biol. 1995; 254: 761-770Crossref PubMed Scopus (159) Google Scholar) and the analysis programs of GROMACS. Pairwise superposition has been performed using the program ProFit (A. C. R. Martin; www.bioinf.org.uk/software/profit/). Apo-cl-BABP NMR Assignment and Structure Calculation—Recombinant cl-BABP has been characterized by 1H, 13C, and 15N NMR. The choice of working at pH 7.0 was dictated by the need to perform structural and dynamic comparisons with the protein in its holo-form at neutral pH. Backbone assignment, performed by a combination of classical three-dimensional NMR experiments, was not straightforward especially for the C-terminal region of the protein corresponding to strands F-I. In this region, breaks in the process of assignment were caused by missing correlations due either to fast exchange of amide protons with solvent and/or to conformational exchange (see below). It was therefore necessary to combine the standard three-dimensional backbone assignment strategy with the sequential assignment strategy. Three-dimensional 1H-15N TOCSY/NOESY, performed at pH 5.6, guided the assignment of those amide resonances in fast exchange with solvent at pH 7.0. In this way the assignment was possible for all but six residues, namely Met73, Val90, Ser93, Lys95,Glu99, and Gln100, located in a region of the protein mostly affected by conformational exchange, as revealed by 15 N relaxation analysis (see below). The 1H, 13C, and 15N assignments of apo-cl-BABP have been deposited in the BioMagResBank (entry code 6642). Three-dimensional 1H-15N TOCSY/NOESY spectra obtained at pH 5.6 revealed the presence of double peaks for several residues, and unambiguous assignment was possible for Ser3 (A strand), Gly44 (BC loop), Phe47 (C strand), Asp74 (EF loop), Ala85 (FG loop), Leu89 (G strand), and Gly 104(HI loop). The small difference in chemical shift of major and minor peaks of ∼20-120 Hz indicated a time scale of exchange of the order of 0.001-0.01 s. These double peaks provide an indication of slow exchange processes affecting the protein backbone. Only totally unambiguous restraints, i.e. those correlations that were not affected by overlap in any spectra, were used for structural calculation. In this way a set of 1000 nonredundant NOEs was supplemented as follows: (i) by 26 distance restraints for 13 backbone hydrogen bonds defined on the basis of deuterium hydrogen exchange studies (data not shown), and (ii) by 48 φ angle constraints derived from Jhn-ha coupling constants. It should be stressed that this protein is highly flexible, as revealed both by H/D exchange and relaxation measurements, and several residues did not exhibit long range NOE correlations (see below). The superposition of the 10 best NMR structures, as obtained after DYANA molecular dynamics simulations followed by energy minimization, reported in Fig. 2, affords an r.m.s.d. backbone (3-125) value of 2.02 ± 0.26 Å. The structural quality of the minimized structures was examined with the PROCHECK-NMR (35Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1096) Google Scholar). Analysis of the backbone dihedral angles showed that 95% of all non-glycine and non-proline residues in apo-cl-BABP fall within the additional allowed regions
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