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• W2017030114 abstract "The solution structure of trimeric Escherichia coli enzyme IIAChb (34 kDa), a component of the N,N′-diacetylchitobiose/lactose branch of the phosphotransferase signal transduction system, has been determined by NMR spectroscopy. Backbone residual dipolar couplings were used to provide long range orientational restraints, and long range (|i - j| ≥ 5 residues) nuclear Overhauser enhancement restraints were derived exclusively from samples in which at least one subunit was 15N/13C/2H/(Val-Leu-Ile)-methyl-protonated. Each subunit consists of a three-helix bundle. Hydrophobic residues lining helix 3 of each subunit are largely responsible for the formation of a parallel coiled-coil trimer. The active site histidines (His-89 from each subunit) are located in three symmetrically placed deep crevices located at the interface of two adjacent subunits (A and C, C and B, and B and A). Partially shielded from bulk solvent, structural modeling suggests that phosphorylated His-89 is stabilized by electrostatic interactions with the side chains of His-93 from the same subunit and Gln-91 from the adjacent subunit. Comparison with the x-ray structure of Lactobacillus lactis IIALac reveals some substantial structural differences, particularly in regard to helix 3, which exhibits a 40° kink in IIALacversus a 7° bend in IIAChb. This is associated with the presence of an unusually large (230-Å3) buried hydrophobic cavity at the trimer interface in IIALac that is reduced to only 45 Å3 in IIAChb. The solution structure of trimeric Escherichia coli enzyme IIAChb (34 kDa), a component of the N,N′-diacetylchitobiose/lactose branch of the phosphotransferase signal transduction system, has been determined by NMR spectroscopy. Backbone residual dipolar couplings were used to provide long range orientational restraints, and long range (|i - j| ≥ 5 residues) nuclear Overhauser enhancement restraints were derived exclusively from samples in which at least one subunit was 15N/13C/2H/(Val-Leu-Ile)-methyl-protonated. Each subunit consists of a three-helix bundle. Hydrophobic residues lining helix 3 of each subunit are largely responsible for the formation of a parallel coiled-coil trimer. The active site histidines (His-89 from each subunit) are located in three symmetrically placed deep crevices located at the interface of two adjacent subunits (A and C, C and B, and B and A). Partially shielded from bulk solvent, structural modeling suggests that phosphorylated His-89 is stabilized by electrostatic interactions with the side chains of His-93 from the same subunit and Gln-91 from the adjacent subunit. Comparison with the x-ray structure of Lactobacillus lactis IIALac reveals some substantial structural differences, particularly in regard to helix 3, which exhibits a 40° kink in IIALacversus a 7° bend in IIAChb. This is associated with the presence of an unusually large (230-Å3) buried hydrophobic cavity at the trimer interface in IIALac that is reduced to only 45 Å3 in IIAChb. The bacterial phosphotransferase system (PTS) 1The abbreviations used are: PTS, phosphotransferase system; Chb, N,N′-diacetylchitobiose; HPr, histidine-containing phosphocarrier protein; IIAChb, IIBChb, and IICChb, A, B, and C domains, respectively, of the N,N′-diacetylchitobiose transporter IIChb; IIAChb*, double mutant of IIAChb comprising a 13-residue deletion at the N-terminus and an Asp to Leu mutation at position 92 (of the double mutant); NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence; TROSY, transverse relaxation optimized spectroscopy; r.m.s., root mean square. is a prototypical signal transduction network in which translocation of sugars across the cytoplasmic membrane is coupled to phosphorylation (1Kundig W. Ghosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (329) Google Scholar, 2Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 3Robillard G.T. Broos J. Biochim. Biophys. Acta. 1999; 1422: 73-104Crossref PubMed Scopus (97) Google Scholar, 4Siebold C. Flükiger K. Beutler R. Erni B. FEBS Lett. 2001; 504: 104-111Crossref PubMed Scopus (93) Google Scholar). There are four branches of the pathway corresponding to four classes of enzymes II, glucose, mannose, mannitol, and lactose/chitobiose, which bear no sequence similarity to one another and for the most part no structural similarity either (2Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 3Robillard G.T. Broos J. Biochim. Biophys. Acta. 1999; 1422: 73-104Crossref PubMed Scopus (97) Google Scholar, 4Siebold C. Flükiger K. Beutler R. Erni B. FEBS Lett. 2001; 504: 104-111Crossref PubMed Scopus (93) Google Scholar). The initial steps of the PTS are common to all branches of the pathway and involve the transfer of a phosphoryl group from phosphoenolpyruvate to the histidine phosphocarrier protein, HPr, via the intermediary enzyme I. Subsequently the phosphoryl group is transferred to sugar-specific enzymes II. Each enzyme II consists of two cytoplasmic domains, IIA and IIB, and a transmembrane sugar permease IIC, which may or may not be covalently linked. IIA accepts the phosphoryl group from HPr and donates it to IIB; IIC catalyzes the transport of the sugar across the membrane and its phosphorylation by IIB. Structures of many of the individual cytoplasmic components of the PTS have been solved by either NMR (5Wittekind M. Rajgopal P. Cranchini B.R. Reizer J. Saier M.H. Klevit R.E. Protein Sci. 1992; 1: 1363-1376Crossref PubMed Scopus (63) Google Scholar, 6Kalbitzer H.R. Hengstenberg W. Eur. J. Biochem. 1993; 216: 205-214Crossref PubMed Scopus (42) Google Scholar, 7van Nuland N.A.J. Hangyi I.W. van Shaik R.C. Berendsen H.J.C. van Gunsteren W.F. Scheek R.M. Robillard G.T. J. Mol. Biol. 1994; 237: 544-559Crossref PubMed Scopus (73) Google Scholar, 8Ab E. Schuurman-Wolters G.K. Reizer J. Saier M.H. Dijkstra M. Scheek R.M. Robillard G.T. Protein Sci. 1997; 6: 304-314Crossref PubMed Scopus (25) Google Scholar, 9Ab E. Shuurman-Walters G.K. Nijlant D. Dijkstra K. Saier M.H. Robillard G.T. Scheek R.M. J. Mol. Biol. 2001; 308: 993-1009Crossref PubMed Scopus (22) Google Scholar, 10Garrett D.S. Seok Y.-J. Liao D.-I. Peterkofsky A. Gronenborn A.M. Clore G.M. Biochemistry. 1997; 36: 2517-2530Crossref PubMed Scopus (152) Google Scholar, 11Legler P.M. Cai M. Peterkofsky A. Clore G.M. J. Biol. Chem. 2004; 279: 39115-39121Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) or crystallography (12Liao D.-I. Kapadia G. Reddy P. Saier M.H. Reizer J. Herzberg O. Biochemistry. 1991; 30: 9538-9594Google Scholar, 13Worthylake D. Meadow N.D. Roseman S. Liao D.-I. Herzberg O. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10383-10386Crossref Scopus (88) Google Scholar, 14Herzberg O. Reddy P. Sutrina S. Saier M.H. Reizer J. Kapadia G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2499-2503Crossref PubMed Scopus (122) Google Scholar, 15Jia Z. Quail J.W. Waygood E.B. Delbaere L.T. J. Biol. Chem. 1993; 268: 22490-22501Abstract Full Text PDF PubMed Google Scholar, 16Liao D.-I. Silverton E. Seok Y.-J. Lee B.R. Peterkofsky A. Davies D.R. Structure. 1996; 4: 861-872Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Nunn R.S. Markovic-Housley Z. Genovesio-Taverne G. Flükiger K. Rizkallah P.J. Jansonius J.N. Schirmer T. Erni B. J. Mol. Biol. 1996; 259: 502-511Crossref PubMed Scopus (61) Google Scholar, 18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 19Van Montfort R.L. Pijning T. Kalk K.H. Reizer J. Saier M.J. Thunnissen M.M. Robillard G.T. Dijkstra B.W. Structure (Lond.). 1997; 5: 217-225Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 20Van Montfort R.L. Pijning T. Kalk K.H. Hangyi I.W. Kouwijzer M.L.C.E. Robillard G.T. Dijkstra B.W. Structure (Lond.). 1998; 6: 377-388Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 21Schauder S. Nunn R.S. Lanz R. Erni B. Schirmer T. J. Mol. Biol. 1998; 276: 591-602Crossref PubMed Scopus (38) Google Scholar, 22Orniss G.L. Erni B. Schirmer T. J. Mol. Biol. 2003; 327: 1111-1119Crossref PubMed Scopus (17) Google Scholar), and more recently we have reported the solution NMR structures of a number of protein-protein complexes of the PTS (23Garrett D.S. Seok Y.-J. Peterkofsky A. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1999; 6: 166-173Crossref PubMed Scopus (211) Google Scholar, 24Wang G. Louis J.M. Sondej M. Seok Y.-J. Peterkofsky A. Clore G.M. EMBO J. 2000; 19: 5635-5649Crossref PubMed Scopus (106) Google Scholar, 25Cornilescu G. Lee B.R. Cornilescu C. Wang G. Peterkofsky A. Clore G.M. J. Biol. Chem. 2002; 277: 42289-42298Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Cai M. Williams D.C. Wang G. Lee B.R. Peterkofsky A. Clore G.M. J. Biol. Chem. 2003; 278: 25191-25206Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). As part of our continuing structural work on the PTS, we present the solution structure of Escherichia coli N,N′-diacetylchitobiose-specific enzyme IIA (IIAChb) using multidimensional NMR spectroscopy. The E. coli N,N′-diacetylchitobiose-specific enzymes II (IIChb) are part of the lactose/chitobiose branch of the PTS (27Keyhani N.O. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14367-14371Crossref PubMed Scopus (86) Google Scholar). The A, B, and C components of IIChb are encoded by a single operon and expressed as individual proteins (27Keyhani N.O. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14367-14371Crossref PubMed Scopus (86) Google Scholar). NMR (8Ab E. Schuurman-Wolters G.K. Reizer J. Saier M.H. Dijkstra M. Scheek R.M. Robillard G.T. Protein Sci. 1997; 6: 304-314Crossref PubMed Scopus (25) Google Scholar, 9Ab E. Shuurman-Walters G.K. Nijlant D. Dijkstra K. Saier M.H. Robillard G.T. Scheek R.M. J. Mol. Biol. 2001; 308: 993-1009Crossref PubMed Scopus (22) Google Scholar) and crystal structures of E. coli IIBChb have been solved and bear surprising similarity to the structure of IIBMannitol (11Legler P.M. Cai M. Peterkofsky A. Clore G.M. J. Biol. Chem. 2004; 279: 39115-39121Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), as well as to that of the low molecular weight eukaryotic protein tyrosine phosphatases (28Zhang M. Van Etten R.L. Stauffacher C.V. Biochemistry. 1994; 33: 11097-11105Crossref PubMed Scopus (119) Google Scholar, 29Su X.-D. Taddei N. Stefani M. Ramponi G. Nordlund P. Nature. 1994; 370: 575-578Crossref PubMed Scopus (205) Google Scholar), despite the absence of any significant sequence identity. No structure, however, has been determined as yet for E. coli IIAChb. The analogous system in Lactobacillus and Staphylococcus comprises the lactose-specific enzymes II (IILac) (30Hays J.B. Simoni R.D. Roseman S. J. Biol. Chem. 1973; 248: 941-956Abstract Full Text PDF PubMed Google Scholar, 31de Vos W.M. Boerrigter I. van Rooyen R.J. Reiche B. Hengstenberg W. J. Biol. Chem. 1990; 265: 22554-22560Abstract Full Text PDF PubMed Google Scholar). The crystal structure of IIALac from Lactobacillus lactis has been solved and been shown to consist of a symmetric homotrimer (18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar): each subunit comprises three antiparallel helices; the trimer interface consists of a parallel coiled-coil formed by the C-terminal helix from each subunit; and the trimer interface is partially stabilized by a metal ion coordinated to the side chains of three buried, symmetry-related, aspartate residues, one from each subunit. The crystal structure of IIALac, however, displays a highly unusual feature in the form of a very large (230-Å3), completely buried cavity at the trimer interface that is associated with the presence of a ∼40° kink in helix 3 and accommodates the heavy atom derivative trimethyl lead acetate (18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). E. coli IIAChb and L. lactis IIALac share 35% sequence identity and 63% amino acid similarity with no gaps or insertions (Fig. 1). In addition, binding of divalent cations to E. coli IIAChb significantly enhances its thermostability relative to the apoform, increasing in the order Mg2+, Cu2+, and Ni2+ (33Keyhani N.O. Boudker O. Roseman S. J. Biol. Chem. 2000; 275: 33091-33101Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). It has also been reported, on the basis of analytical ultracentrifugation data, that E. coli IIAChb is dimeric in solution (34Keyhani N.O. Rodgers M.E. Demeler B. Hansen J.C. Roseman S. J. Biol. Chem. 2000; 275: 33110-33115Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) in clear contrast to the trimeric state of IIALac (18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Since the three symmetrically related active site histidines in IIALac are located in a crevice formed by the interface of two adjacent subunits (18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), this result would imply that the surface topology of the active sites and the spatial relationships of side chains within the active sites are very different in IIALac and IIAChb. If true, this would be highly unexpected given the close sequence and functional relationship between IIALac and IIAChb. Intrigued by this discrepancy, we initiated our own investigation of the oligomerization state and three-dimensional solution structure of E. coli IIAChb. Protein Purification and Mutagenesis—The plasmid for wild-type E. coli IIAChb was a kind gift from Dr. Saul Roseman (The Johns Hopkins University, Baltimore, MD). The plasmid was propagated in TOP10 cells, and IIAChb was expressed in BL21-Star cells (Invitrogen) at 37 °C. After induction with isopropyl β-d-thiogalactopyranoside, cells were harvested and microfluidized in 10 mm Tris·HCl, pH 7.5, buffer. The protein was purified using a DEAE-Sepharose anion-exchange column followed by a preparative Superdex-75 size exclusion column (Amersham Biosciences). IIAChb was denatured with 6 m guanidine HCl solution (pretreated with Chelex; Bio-Rad) at pH 3 and incubated at 37 °C overnight to remove residual phosphoryl groups on the protein. The pH of the denatured protein solution was then raised by the addition of 1 m Tris·HCl buffer. The protein solution was then dialyzed against either 10 mm sodium phosphate buffer (pH 6.5) or 10 mm Tris·HCl buffer (pH 8) containing 100 μm divalent cation to remove guanidine HCl and refold the protein; seven metal ions, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+, were tested individually, yielding seven different samples. Excess metal ions in each metal-loaded protein sample were washed away by extensive buffer exchange in a concentrator, and the protein was further concentrated. The N-terminal deletion mutation (IIAChb-NΔ13) in which the first 13 residues were removed and the subsequent point mutation, IIAChb-NΔ13/D92L, were introduced using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The primers were designed according to the manufacturer's instructions. The sequences of the two mutants (IIAChb-NΔ13 and IIAChb-NΔ13/D92L) were confirmed by DNA sequencing (Davis Sequencing, Davis, CA). The two mutant proteins were purified using the same procedure described above for the wild-type protein. Typically 50 mg of protein were obtained from a 1-liter culture. The masses of the unlabeled proteins (wild type, NΔ13, and NΔ13/D92L) were confirmed by electrospray ionization mass spectrometry. The double mutant NΔ13/D92L is referred to hereafter as IIAChb*. (Ile/Leu/Val)-methyl-protonated and otherwise fully deuterated IIAChb-NΔ13/D92L (abbreviated as ILV-IIAChb*) was expressed based on the protocol described previously (35Goto N.K. Gardner K.H. Mueller G.A. Willis R.C. Kay L.E. J. Biomol. NMR. 1999; 13: 369-374Crossref PubMed Scopus (443) Google Scholar). Briefly cells were grown in M9 minimal medium prepared in D2 O with [2H7, 13C6]glucose and [15N]NH4Cl as the carbon and nitrogen sources, respectively. 80 mg of α-[13C5,3-2H1]ketoisovalerate and 50 mg of α-[13C4,3,3-2H2]ketobutyrate (Cambridge Isotopes, Andover, MA) were added into 1 liter of culture 45 min prior to induction at an A600 nm of ∼0.6. After induction with isopropyl β-d-thiogalactopyranoside, cells were grown under vigorous shaking for another 4 h before harvesting. Purified in H2O buffer, the protein sample carries protons only at backbone and side-chain amides, γ-methyls of Val residues, and δ-methyl(s) of Ile/Leu residues. Light Scattering—Static light-scattering data were collected using analytical size exclusion chromatography on a Superdex-75 column (Amersham Biosciences) in tandem with DAWN EOS light scattering and refractive index detectors (Wyatt Technology, Santa Barbara, CA). 100 μl of 75 μg of protein was applied to the pre-equilibrated Superdex-75 column at a flow rate of 0.5 ml/min at room temperature (20 °C). The running buffer consisted of either 10 mm sodium phosphate buffer, pH 6.5, or 10 mm Tris·HCl buffer, pH 7.5, containing 0.02% NaN3, 1 mm methionine, and 300 mm NaCl. The protein elution profile was monitored by the refractive index detector, and light-scattering measurements were made every 4 μl for a total elution volume of 20 ml. The data were analyzed using the manufacturer's proprietary software. The translational diffusion coefficient of the IIAChb-NΔ13/D92L double mutant (IIAChb*) was determined from autocorrelation analysis of quasielastically scattered light. Autocorrelation functions were collected on a BI-9000 AT autocorrelator (Brookhaven Instruments, Long Island, NY) at an angle of 90° with sampling times ranging from 0.5 μs to 10 ms. The diffusion coefficient, D20,w, was derived from autocorrelation functions of 10 independent measurements using the software provided by Brookhaven Instruments. The corresponding Stokes radius, Rs, was calculated using the equation Rs = kT/6πηD20,w where η represents the solvent viscosity, T is the absolute temperature, and k is the Boltzmann constant. The predicted translational diffusion coefficient and Stokes radius for the crystal structure of L. lactis IIALac (18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) were calculated using the program HYDROPRO (36Garcia de la Torre J. Huertas M.L. Carrasco B. Biophys. J. 2000; 78: 719-730Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar). NMR Data Collection and Analysis—All NMR samples were prepared in 10 mm sodium phosphate buffer, pH 6.5, containing 0.02% NaN3 and 1 mm methionine. NMR spectra were collected at 30 °C on Bruker DMX500, DMX600, DRX600, DMX750, and DRX800 spectrometers equipped either with x,y,z-shielded gradient triple resonance probes or z-shielded gradient triple resonance cryoprobes. Spectra were processed with the NMRPipe/nmrDraw suite (37Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfiefer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed using the PIPP/CAPP/STAPP package (38Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Sequential resonance assignments were derived from analysis of transverse relaxation optimized (TROSY)-based triple resonance three-dimensional NMR experiments (39Clore G.M. Gronenborn A.M. Science. 1991; 252: 1390-1399Crossref PubMed Scopus (421) Google Scholar, 40Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (796) Google Scholar, 41Yang D.W. Kay L.E. J. Biomol. NMR. 1999; 14: 273-276Crossref Scopus (30) Google Scholar, 42Clore G.M. Gronenborn A.M. Trends Biotechnol. 1998; 16: 22-34Abstract Full Text PDF PubMed Scopus (228) Google Scholar), including HNCO, HN(CO)CA, HNCA, HNCB, and HN(CO)CB recorded on the ILV-IIAChb* sample. Nearly complete side-chain assignments were obtained from analysis of a three-dimensional HCCH-TOCSY experiment recorded on a 13C, 15N-labeled IIAChb* sample. A triple resonance CBCA(CO)NH was also recorded on the 13C, 15N-labeled IIAChb* sample to obtain 13Cα/13Cβ chemical shifts free of the offsets resulting from perdeuteration. Backbone φ/Ψ torsion angle restraints were derived from backbone 1H, 15N, and 13C chemical shifts using the program TALOS (43Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). Side-chain torsion angle restraints were derived from 3JNCγ, 3JC′Cγ, and 3JCαCδ coupling constants measured using quantitative J correlation experiments (44Bax A. Vuister G.W. Grzesiek S. Delaglio F. Wang A.C. Tschudin R. Zhu G. Methods Enzymol. 1994; 239: 79-105Crossref PubMed Scopus (381) Google Scholar). Intersubunit nuclear Overhauser enhancements (NOEs) were obtained from a three-dimensional 13C-separated/12C-filtered NOE spectrum collected on a sample comprising a 1:1 mixture of unlabeled IIAChb* and labeled ILV-IIAChb* (42Clore G.M. Gronenborn A.M. Trends Biotechnol. 1998; 16: 22-34Abstract Full Text PDF PubMed Scopus (228) Google Scholar). To ensure complete mixing of the subunits, the 1:1 mixture of the labeled and unlabeled proteins was denatured in 6 m guanidine HCl and then refolded into the NMR buffer (10 mm sodium phosphate buffer, pH 6.5, 0.02% NaN3, and 1 mm methionine). Intramolecular long range NOEs were obtained from four-dimensional 13C/15N-separated and 13C/13C-separated NOE spectra collected on the ILV-IIAChb* sample. As methyl-methyl NOE interactions in the unfiltered NOE spectra contain both intra- and intermolecular information, care was taken to ensure that assigned intramolecular NOE cross-peaks did not appear in the three-dimensional 13C-separated/12C-filtered NOE spectrum. NOEs involving backbone amide protons were obtained from a three-dimensional 15N-separated NOE spectrum. Backbone 1DNH, 1DNC′, and 1DCαC′ residual dipolar couplings were obtained from the difference between the 1J scalar couplings measured in dilute liquid crystalline (15 mg/ml phage pf1 (45Clore G.M. Starich M.R. Gronneborn A.M. J. Am. Chem. Soc. 1998; 120: 10571-10572Crossref Scopus (323) Google Scholar, 46Hansen M.R. Hanson P. Pardi A. Methods Enzymol. 2000; 317: 220-240Crossref PubMed Google Scholar)) and isotropic (water) media. 1JNH and 1JNC′ couplings were measured from the splittings in three-dimensional HNCO-TROSY-based experiments (47Yang D. Venters R.A. Mueller G.A. Choy W.Y. Kay L.E. J. Biomol. NMR. 1999; 14: 333-343Crossref Scopus (133) Google Scholar), and 1JCαC′ couplings were derived from an intensity-modulated HN(CO)CATROSY experiment (48Jaroniec C.P. Ulmer T.S. Bax A. J. Biomol. NMR. 2004; 30: 181-194Crossref PubMed Scopus (25) Google Scholar). Structure Calculation—NOE-derived interproton distance restraints were classified into distance ranges of 1.8–2.7, 1.8–3.3, 1.8–5.0, and 1.8–6 Å, corresponding to strong, medium, weak, and very weak NOE cross-peak intensities, respectively. An additional 0.5 Å was added to the upper distance bound of distance restraints involving methyl groups (0.5 Å per methyl group), and 0.2 Å was added to the upper bounds for strong and medium NOE restraints involving amide protons. Nonstereospecifically assigned methyl protons and ambiguous intermolecular NOEs were represented by a (Σr-6)-1/6 sum. The error ranges used for the torsion angle restraints (which are represented by square-well potentials) are ±20° for the backbone φ/Ψ angles within helical regions and ±20° for χ1 and ±30° for χ2 side-chain torsion angles. χ1 restraints for aliphatic side chains that are not in the t rotamer and not experiencing rotamer averaging (3JCγN < 1.0 Hz) were set to 0 ± 80°. Structures were calculated using a well established protocol (49Nilges M. Gronenborn A.M. Brünger A.T. Clore G.M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (516) Google Scholar, 50Garrett D.S. Kuszewski J. Hancock T.J. Lodi P.J. Vuister G.W. Gronenborn A.M. Clore G.M. J. Magn. Reson. B. 1994; 104: 99-103Crossref PubMed Scopus (133) Google Scholar, 51Kuszewski J. Qin J. Gronenborn A.M. Clore G.M. J. Magn. Reson. B. 1995; 106: 92-96Crossref PubMed Scopus (190) Google Scholar, 52Clore G.M. Gronenborn A.M. Tjandra N. J. Magn. Reson. 1988; 131: 159-162Crossref Scopus (278) Google Scholar) from the experimental NMR restraints by simulated annealing in torsion angle space (53Schwieters C.D. Clore G.M. J. Magn. Reson. 2001; 152: 288-302Crossref PubMed Scopus (164) Google Scholar) using the program Xplor-NIH (54Schwieters C.D. Kuszewski J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1882) Google Scholar). The coordinates of the three subunits were restrained to their average positions (after best fitting) by a non-crystallographic symmetry restraint. The non-bonded contacts in the target function were represented by a quartic van der Waals repulsion term (49Nilges M. Gronenborn A.M. Brünger A.T. Clore G.M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (516) Google Scholar) supplemented by multidimensional torsion angle (55Clore G.M. Kuszewski J. J. Am. Chem. Soc. 2002; 124: 2866-2867Crossref PubMed Scopus (74) Google Scholar) and backbone hydrogen bonding (56Grishaev A. Bax A. J. Am. Chem. Soc. 2004; 126: 7281-7292Crossref PubMed Scopus (100) Google Scholar) data base potentials of mean force. A radius of gyration restraint was used to ensure optimal packing (57Kuszewski J. Gronenborn A.M. Clore G.M. J. Am. Chem. Soc. 1999; 121: 2337-2338Crossref Scopus (220) Google Scholar). Structure figures were generated with the programs VMD-XPLOR (58Schwieters C.D. Clore G.M. J. Magn. Reson. 2001; 149: 239-244Crossref PubMed Scopus (111) Google Scholar) and Pymol (59DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Reweighted atomic density probability maps were calculated from the structure ensemble as described previously (60Schwieters C.D. Clore G.M. J. Biomol. NMR. 2002; 23: 221-225Crossref PubMed Scopus (61) Google Scholar). Modeling of the phosphorylated state was carried out by manually docking a phosphoryl group in the vicinity of the Nϵ-1 atom of the active site His-89 of the restrained regularized mean structure in VMD-XPLOR followed by regularization using Xplor-NIH, keeping all coordinates fixed with the exception of the side chain of His-89 and the phosphoryl group. Engineering a Monodisperse Trimer—1H-15N heteronuclear single quantum coherence (HSQC) correlation spectra of 15N-labeled IIAChb in both the apoform and loaded with diverse divalent cations, including Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+, are all broad and poorly dispersed, indicative of chemical exchange between various states. Of these various samples, Ni2+-charged IIAChb displayed the best spectrum (Fig. 2A), suggesting slightly more favorable exchange dynamics, consistent with the prior observation that Ni2+ has the largest effect in enhancing the thermostability of IIAChb (33Keyhani N.O. Boudker O. Roseman S. J. Biol. Chem. 2000; 275: 33091-33101Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Ni2+-charged IIAChb was then subjected to light-scattering analysis. The intensity of light scattered by a molecule is directly proportional to molar mass; thus static light scattering used in tandem with analytical size exclusion chromatography can measure the average molecular weight of the eluted fractions on the fly. Light-scattering analysis reveals that wildtype Ni2+-IIAChb is a mixture of many species of different molecular masses ranging from 35 to 70 kDa, and there is no predominant species of defined molecular weight in the mixture (Fig. 3A, blue symbols). This result fully accounts for the poor quality of the 1H-15N correlation spectrum of Ni2+-IIAChb (Fig. 2A). Significantly, even at the leading edge of the gel filtration peak where the protein concentration is only ∼140 nm, the molecular mass never falls below 3 times the monomer molecular mass (12,747.7 Da), which is the molecular mass of a trimer. Further, as increasing concentration promotes higher order oligomerization, our light-scattering results are not consistent with the previous report that IIAChb behaves as a dimer even at a concentration of 30 μm or an optical density of 1 at 230 nm (34Keyhani N.O. Rodgers M.E. Demeler B. Hansen J.C. Roseman S. J. Biol. Chem. 2000; 275: 33110-33115Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). To obtain a homogeneous and monodisperse sample suitable for structural studies, we constructed a series of IIAChb variants using rational mutagenesis. We first noted that (a) the N-terminal 11 residues of IIAChb are not present in L. lactis IIALac (Fig. 1), (b) the N-terminal two residues of IIALac have high B-factors in the crystal structure (18Sliz P. Engelmann R. Hengstenberg W. Pai E.F. Structure (Lond.). 1997; 5: 775-788Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and (c) the secondary structure of the first 13 residues of IIAChb is predicted to be unstructured (PredictProtein, cubic.bioc.columbia.edu/predictprotein/). On this basis we constructed a deletion mutant of IIAChb lacking the N-terminal 13 residues, IIAChb-NΔ13 (Fig. 1). The light-scattering profile for Ni2+-loaded IIAChb-NΔ13 is also a mixture of different molecular mass species ranging from 35 to 70 kDa (Fig. 3A, green symbols). U" @default.
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• W2017030114 title "Solution Structure of Enzyme IIAChitobiose from the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System" @default.
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