FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2110685170> ?p ?o ?g. }

• W2110685170 endingPage "16328" @default.
• W2110685170 startingPage "16320" @default.
• W2110685170 abstract "The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2–50 were found to form a compact subdomain containing three short β-strands and three α-helices, folded to form a βααββα motif with the three helices packed on the same side of a small twisted β-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51–97 are less conserved and do not appear to form a regular structure, whereas residues 98–157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2. The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2–50 were found to form a compact subdomain containing three short β-strands and three α-helices, folded to form a βααββα motif with the three helices packed on the same side of a small twisted β-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51–97 are less conserved and do not appear to form a regular structure, whereas residues 98–157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2. initiation factor root mean square deviation stem contact matrix-assisted laser desorption/ionization time-of-flight chemical shift index protein data bank The initiation step of protein biosynthesis is rate-limiting and hence an important point of regulation. In bacteria, translation initiation is promoted by three protein factors: IF1,1 IF2, and IF3. These protein factors are essential in ultimately assembling the 30 S and 50 S subunits of the ribosome, the initiator fMet-tRNAfMet, and the translation initiation region of the mRNA, thereby forming the functional 70 S initiation complex. Bacterial translation initiation factor IF2 ensures correct binding of fMet-tRNAfMet to the P-site of the 30 S ribosomal subunit. Subsequently, the 50 S ribosomal subunit joins the 30 S initiation complex aided by IF2. GTP bound to the factor is hydrolyzed in a ribosome-dependent reaction. IF2 is then released from the ribosome leaving fMet-tRNAfMet in the ribosomal P-site. Recent reviews of the translation initiation process are provided in Refs. 1Ramakrishnan V. Cell. 2002; 108: 557-572Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 2Moreno J.M. Sørensen H.P. Mortensen K.K. Sperling-Petersen H.U. IUBMB Life. 2000; 50: 347-354Crossref PubMed Google Scholar, 3Gualerzi C.O. Brandi L. Caserta E. Teana A.L. Spurio R. Tomsic J. Pon C.L. Garret R.A. Douthwaite S.R. Liljas A. Matheson A.T. Moore P.B. Noller H.F. The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. ASM Press, Washington, D. C.2000: 477-494Google Scholar, 4Boelens R. Gualerzi C.O. Curr. Protein Pept. Sci. 2002; 3: 107-119Crossref PubMed Scopus (65) Google Scholar. The primary structure of initiation factor IF2 from different organisms can be divided into distinct regions based on interspecies amino acid sequence homology (5Steffensen S.A. Poulsen A.B. Mortensen K.K. Sperling-Petersen H.U. FEBS Lett. 1997; 419: 281-284Crossref PubMed Scopus (19) Google Scholar), as shown in Fig.1. The C-terminal region of the protein is highly conserved among species. This part has several functions including a binding site for fMet-tRNAfMet, a site for binding and hydrolysis of GTP, and a site for interaction with IF1 (Ref. 6Sørensen H.P. Hedegaard J. Sperling-Petersen H.U. Mortensen K.K. IUBMB Life. 2001; 51: 321-327Crossref PubMed Scopus (19) Google Scholar and references therein). Sequence homologues of IF2 have been found in Archaea and eukaryotes, where the factor is referred to as aIF5B and eIF5B, respectively (7Kyrpides N.C. Woese C.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 224-228Crossref PubMed Scopus (201) Google Scholar). aIF5B and eIF5B have some functional similarity to bacterial IF2; each of these proteins has GTPase activity, and promotes ribosomal subunit joining and probably interaction with Met-tRNAiMet (8Choi S.K. Lee J.H. Zoll W.L. Merrick W.C. Dever T.E. Science. 1998; 280: 1757-1760Crossref PubMed Scopus (113) Google Scholar, 9Pestova T.V. Lomakin I.B. Lee J.H. Choi S.K. Dever T.E. Hellen C.U. Nature. 2000; 403: 332-335Crossref PubMed Scopus (301) Google Scholar). The crystal structure of archaeal aIF5B from Methanobacterium thermoautothrophicum has been solved (10Roll-Mecak A. Cao C. Dever T.E. Burley S.K. Cell. 2000; 103: 781-792Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar); sequence homology predicts a similar structure for the conserved C terminus of IF2 from bacteria. The structure of the 99-residue Met-tRNAiMet binding domain of IF2 from the bacterium Bacillus stearothermophilus has been solved using NMR methods (11Meunier S. Spurio R. Czisch M. Wechselberger R. Guenneugues M. Gualerzi C.O. Boelens R. EMBO J. 2000; 19: 1918-1926Crossref PubMed Scopus (63) Google Scholar). There is, however, no previously reported crystallographic or NMR structural study of Domain I at the N terminus of bacterial IF2 (Fig. 1), which is the subject of the present work. Bacterial IF2 is encoded by the infB gene, which inEscherichia coli encodes three forms of IF2: IF2–1, -2, and -3, of molecular masses 97.3, 79.9, and 78.8 kDa, respectively (12Nyengaard N.R. Mortensen K.K. Lassen S.F. Hershey J.W. Sperling-Petersen H.U. Biochem. Biophys. Res. Commun. 1991; 181: 1572-1579Crossref PubMed Scopus (25) Google Scholar). The expression of IF2–2 and IF2–3 in E. coli is by tandem translation of the intact infB mRNA, and not by translation of post-transcriptionally truncated mRNA. Hence, the three different forms of IF2 have identical C termini (13Mortensen K.K. Hajnsdorf E. Regnier P. Sperling-Petersen H.U. Biochem. Biophys. Res. Commun. 1995; 214: 1254-1259Crossref PubMed Scopus (7) Google Scholar). The presence of both the large and smaller forms is required for optimal growth of E. coli. The cellular content of IF2-2 and -3 is close to the level of IF2-1 (14Howe J.G. Hershey J.W. J. Biol. Chem. 1983; 258: 1954-1959Abstract Full Text PDF PubMed Google Scholar, 15Sacerdot C. Vachon G. Laalami S. Morel-Deville F. Cenatiempo Y. Grunberg-Manago M. J. Mol. Biol. 1992; 225: 67-80Crossref PubMed Scopus (43) Google Scholar). The presence of more than one isoform of IF2 is not a phenomenon peculiar to E. coli, but has been found in several other enterobacteria (16Laursen B.S. Steffensen S.A. Hedegaard J. Moreno J.M. Mortensen K.K. Sperling-Petersen H.U. Genes Cells. 2002; 7: 901-910Crossref PubMed Scopus (17) Google Scholar). The N-terminal region of IF2 differs from the C-terminal region in that there is significantly more variability between species in primary structure as well as length. We have previously used sequence data and biochemical experiments to divide the N-terminal region of E. coli IF2 into three separate domains designated Domain I, II, and III (17Mortensen K.K. Kildsgaard J. Moreno J.M. Steffensen S.A. Egebjerg J. Sperling-Petersen H.U. Biochem. Mol. Biol. Int. 1998; 46: 1027-1041PubMed Google Scholar), as illustrated in Fig. 1. A function for the domains in the N-terminal region has been demonstrated in E. coli, where a fragment of IF2 consisting of Domains I and II, but not a fragment consisting of Domain I alone, binds to the 30 S ribosomal subunit (18Moreno J.M. Kildsgaard J. Siwanowicz I. Mortensen K.K. Sperling-Petersen H.U. Biochem. Biophys. Res. Commun. 1998; 252: 465-471Crossref PubMed Scopus (34) Google Scholar,19Moreno J.M. Drskjotersen L. Kristensen J.E. Mortensen K.K. Sperling-Petersen H.U. FEBS Lett. 1999; 455: 130-134Crossref PubMed Scopus (52) Google Scholar). Furthermore, we have recently used a primer extension inhibition assay to identify Domains I-II of E. coli IF2 as an interaction partner for the infB mRNA (16Laursen B.S. Steffensen S.A. Hedegaard J. Moreno J.M. Mortensen K.K. Sperling-Petersen H.U. Genes Cells. 2002; 7: 901-910Crossref PubMed Scopus (17) Google Scholar). The present work describes the results of nuclear magnetic resonance (NMR) and circular dichroism (CD) experiments used to characterize the 18-kDa Domain I of E. coli IF2. A search among structures in the Protein Data Bank revealed that this domain has no significant primary sequence homology to any protein of known structure, and a BLAST search (20Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59416) Google Scholar) of the non-redundant protein sequence data available at the National Center for Biotechnology Information (NCBI) Web site showed that the domain has no significant sequence homologue other than the same domain within IF2 of different species. The fragment of the infB gene encoding the first domain of IF2–1 was amplified by PCR using E. coli K12 as template and primers that included unique restriction sites for XbaI andNdeI for insertion into the pET-15b expression vector (Novagen). DNA sequencing confirmed the insertion of infBinto the vector. The protein was expressed in BL21(DE3) cells (Novagen). Cells were grown in M9 minimal medium supplemented with 100 mg/liter ampicillin. Protein expression was induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside when the cells reached an OD550 of 0.6. Cells from a 1-liter culture were harvested by centrifugation and dissolved in 20 ml of buffer A (50 mm Hepes, pH 7.6, 10 mmMgCl2, 1 mm dithiothreitol, 0.1 mmphenylmethylsulfonyl fluoride, 15 mm NaN3). The solution was passed once through a French pressure cell at 1500 PSI and centrifuged at 30,000 × g for 1 h. The supernatant was loaded on a 40-ml SP Sepharose FF column (Amersham Biosciences), and bound protein was eluted with a 0–200 mmNaCl step gradient. The buffer was changed to buffer A using a Sephadex G25 column (Amersham Biosciences). The pooled fraction was passed through a Source 30Q column, and the unbound protein was loaded on a 20-ml SP Sepharose HP column (Amersham Biosciences). The IF2 Domain I was eluted with a gradient from 0 to 200 mm NaCl over 8 column volumes, yielding 40 mg of pure protein per 1 liter of culture medium. The purified protein was subjected to N-terminal sequencing by Edman degradation, and the protein mass was determined by MALDI-TOF analysis. Samples of protein enriched in 15N or15N and 13C simultaneously were prepared as described above, but cells were grown in M9 minimal medium containing 1.5 g/liter [13C] glucose and/or 0.6 g/liter [15N] ammonium chloride (Cambridge Isotope Laboratories) as sources of carbon and nitrogen, respectively. The circular dichroism spectra were recorded on the UV1 photobiology synchrotron beamline at the Institute for Storage Ring Facilities at Aarhus University, Denmark, using synchroton radiation provided by the ASTRID storage ring. Spectra were recorded in 10 mm phosphate buffer, pH 6.0 using an open 0.01-mm Hellwa suprisil quartz cell. The data were acquired using 5 consecutive scans with 1-nm intervals in the range 180–250 nm. Spectra of each sample were recorded from 5 to 70 °C in 5 °C steps. The sample was allowed to equilibrate at each temperature for 20 min before acquiring the spectra. The data were normalized to a 1 mg/ml concentration in a 1-mm path length cell. NMR spectra were recorded at 20, 30, and 40 °C using a 500 MHz Varian Inova spectrometer equipped with a triple-resonance probe and z-axis pulsed-field gradient. NMR samples typically contained 2–3 mm of the protein and 10 mm sodium phosphate in 90% H2O/10% D2O or 100% D2O solvent at pH 6.0. Backbone resonance assignments were obtained by analyzing HNCA, HNCO, HNCACB, HN(CO)CACB, and HACACBCO spectra, which correlate the backbone protons to the N, Ca, Cb, and CO signals of the same and adjacent amino acid residues. 15N-edited HSQC-TOCSY, 13C-edited HCCH-TOCSY, and two-dimensional 2QF-COSY and TOCSY spectra were used for side-chain resonance assignments. NOE cross-peaks were detected using two-dimensional1H-1H NOESY, three-dimensional15N-resolved 1H-1H HSQC-NOE, and three-dimensional 13C-edited 1H-1H HSQC-NOESY spectra. The 13C-edited1H-1H NOE spectrum was collected in 90% H2O/10%D2O solvent, so that NOE peaks between amide and side-chain protons could be resolved by the chemical shift of a side-chain 13C nucleus. Rapidly exchanging amide protons were identified by comparing 15N-1H correlated spectra obtained with selective excitation versuspresaturation for solvent suppression. Data were processed using either the program NMR-Pipe (21Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11443) Google Scholar) or Felix 1.0 (Hare Research). 1H,15N, and 13C chemical shifts are referenced as recommended by Ref. 22Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2058) Google Scholar, with proton chemical shifts referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 0 ppm. The 0 ppm 13C and 15N reference frequencies were determined by multiplying the 0 ppm 1H reference by 0.251 449 530 and 0.101 329 118, respectively. Structure calculations for IF2 Domain I were performed using the hybrid distance geometry-simulated annealing and energy minimization protocols within the CNS version 1.1 program suite (23Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16947) Google Scholar). Distance restraints were derived from multidimensional NOE spectra. In order to minimize the effects of spin diffusion, as many of the NOE cross-peaks as possible were identified in homonuclear two-dimensional NOE spectra acquired with relatively short mixing times (60 ms); these spectra also offered the best digital resolution. Peaks from these short mixing time spectra were placed into four categories: strong (<3.2 Å), medium (<3.6 Å), weak(< 4.2 Å), and very weak (<4.6 Å). Additional NOE cross-peaks were identified in the three-dimensional 15N- and 13C-edited NOE spectra (60-ms mixing time) and assigned to distance restraints as strong (<5.0 Å), medium (<5.5 Å), weak (<6.3 Å), and very weak (<6.9 Å). A very conservative distance restraint of <7.9 Å was used for NOE cross-peaks identified in spectra obtained with a relatively long mixing time (160 ms), where the effects of spin diffusion are most likely to be present. Pseudoatom corrections were included for NOEs including stereospecifically unassigned methyl protons of Val or Leu, where distances were measured from the center of the two methyl groups, and 2.5 Å was added to the interproton distance. For NOEs involving other methyl groups distances were measured from the center of the methyl group, and 1.0 Å was added to the interproton distance. Stereospecifically unassigned methylene groups were treated the same way, and 0.7 Å was added to the interproton distance. For NOEs involving δ and ε protons of Phe, distances were measured from the central point between the two atoms, and 2.4 Å was added to the interproton distance. For regions of regular α-helix or β-strand structure identified by characteristic NOE patterns and chemical shift indices (CSI) (24Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (933) Google Scholar), backbone torsion angle restraints were included for the ϕ and ψ angles. For β-strands, ϕ and ψ angles were restricted to −120o ± 25o and 150o ± 25o, respectively, and for α-helices both ϕ and ψ angles were restricted to −60o ± 25o. Hydrogen bond restraints were only included for amide protons with relatively slow solvent exchange rates that are also located in regions of regular α-helix or β-sheet structure. Twenty diverse starting structures were generated by subjecting a random coil model to the CNS simulated annealing protocol using only the dihedral angle and hydrogen bond constraints. These structures were then used as starting models for 200 runs of the simulated annealing protocol. Most of the simulated annealing runs resulted in similar structures with similar energies. From this final set of refined models, a set of 20 structures were selected that satisfy the following criteria: 1) their CNS energy term is at or very near the minimum value obtained, 2) there are no interproton distance constraint violations of greater than 0.5 Å, 3) the set of models are a fair representation of the full range of structures that satisfy the NMR-derived restraints while having reasonable molecular geometry, as defined by the CNS energy function. Structural statistics (Table I) were calculated with the assistance of the program PROCHECK-NMR (25Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4362) Google Scholar).Table ISummary of refinement and structural statistics for E. coli IF2 res. 2–50Total number of NOE restraints918 Intraresidue NOEs295 Sequential NOEs (i to i+ 1)227 Medium range NOEs (i toi + 2, 3, 4)215 Long range NOEs181Hydrogen bond restraints15Dihedral angle restraints (φ plus φ)63Number of unique starting structures for simulated annealing20Number of simulated annealing runs, differing in initial trajectories200R.m.s.d. for backbone atoms (Å)0.71R.m.s.d. for all heavy atoms (Å)1.67Number of NOE violations > 0.5ÅnoneNumber1-aGlycine and end residues are not counted. of residues in most favored regions of Ramachandran plot34Number1-aGlycine and end residues are not counted. of residues in additional allowed regions of Ramachandran plot7Number1-aGlycine and end residues are not counted.of residues in generously allowed regions of Ramachandran plot3Number1-aGlycine and end residues are not counted. of residues in disallowed regions of Ramachandran plot1-bThe two residues in disallowed regions of the Ramachandran plot are L7 and D35; these residues are in turn regions where the uncertainty in the structure may account for their presence in disallowed regions.2R.m.s.d. from ideal bond lengths (Å)0.0047 ± 0.0003R.m.s.d. from ideal covalent angles (deg)0.79 ± 0.033R.m.s.d. for improper angles (deg)0.75 ± 0.048Statistics are derived from a set of 20 low-energy structures, a set that is representative of the range of structures that are consistent with the structural constraints.1-a Glycine and end residues are not counted.1-b The two residues in disallowed regions of the Ramachandran plot are L7 and D35; these residues are in turn regions where the uncertainty in the structure may account for their presence in disallowed regions. Open table in a new tab Statistics are derived from a set of 20 low-energy structures, a set that is representative of the range of structures that are consistent with the structural constraints. The 15NT 1 and T 2 relaxation times and the 15N-1H NOE were measured using pulse sequences (26Farrow 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 (2002) Google Scholar) that feature gradient selection and sensitivity enhancement, and pulses for minimizing saturation of the solvent water. Six two-dimensional spectra with relaxation delays of 10, 260, 510, 760, 1010, and 1260 ms were acquired for the T 1relaxation measurements, and six two-dimensional spectra with relaxation delays of 29, 58, 87, 116, 145, and 174 ms were acquired for the T 2 relaxation measurements; in each case the relaxation delay between the acquisition of each free induction decay was 3 s. The spectra for measuring the15N-1H NOE were acquired with either a 5-s delay between each free induction decay or a 1-s delay followed by a 4-s long series of 120o nonselective 1H pulses. The T 1 and T 2 data were fitted to a single exponential decay function of the form I = I0e−t/Td, in which I is the intensity of the signal at time t, I0 is the intensity at time t = 0 and T dis the decay constant T 1 orT 2, respectively. Rotational correlation times and order parameters were calculated using Modelfree 4.0 (27Lillemoen J. Hoffman D.W. J. Mol. Biol. 1998; 281: 539-551Crossref PubMed Scopus (9) Google Scholar) as previously described (28Mandel A.M. Akke M. Palmer III, A.G. J. Mol. Biol. 1995; 246: 144-163Crossref PubMed Scopus (902) Google Scholar). Domain I of E. coli translation initiation factor IF2 was recombinantly expressed, and the purified domain found to be soluble, stable, and well suited for study using biophysical methods. MALDI-TOF mass spectrometry revealed that the N-terminal methionine residue was post-translationally removed from the protein. CD spectra contain features typical of a protein with substantial α-helical content (Fig. 2 A), with characteristic minima at 207 and 222 nm. CD spectra recorded at 30 °C or below look essentially the same, whereas spectra recorded at higher temperatures differ significantly, presumably due to unfolding of the protein (Fig. 2 B). The circular dichroism at 207 nm increases with increasing temperature, consistent with a decrease in helical content. The presence of an isodichroic point in the CD spectra indicates that the unfolding is a two-state process. The protein can be reversibly denatured by heating; a sample heated to 70 °C and then cooled to 20 °C has a CD spectrum that is the same as that recorded before heating of the sample. Two-dimensional NMR spectra acquired at 20 and 30 °C are essentially identical and of excellent quality, with the majority of the resonances being well dispersed, as is typical for a folded protein (Fig.3). However, most NOE cross-peaks were absent in spectra acquired at 40 °C. The NMR and CD data are therefore consistent in indicating that the domain starts to lose structure between 30 and 40 °C.Figure 315 N-1H correlated HSMQC spectrum of Domain I of IF2. The spectrum was acquired at 30 °C in 10 mm phosphate at pH 6. Resonance assignments of the most well resolved cross-peaks are labeled. Chemical shift assignments for the domain of IF2 have been submitted to the BioMagResBank and assigned the accession number BMRB-5624.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An abundance of inter-residue NOE cross-peaks were observed for residues 2–50 of Domain I, consistent with these residues forming a compact globular subdomain. In contrast, residues 51–157 only exhibit NOE cross-peaks between pairs of protons that are relatively close together in the primary sequence; these residues are therefore likely to form a linker region connecting the compact folded structure formed by amino acids 2–50 with the other domains of IF2. The structural details of each of these subdomains within Domain I of IF2 will be discussed in turn. A summary of local NOE patterns and CSI, which define the secondary structure of the protein are given in Fig.4. The structure of residues 2–50 at the N terminus of IF2 was determined from distance constraints derived from observed NOE intensities, and torsion angle and hydrogen bond constraints derived for the regions identified as having regular β-sheet or helical structure. Complete backbone and nearly complete side chain chemical shift assignments were obtained for the 1H, 13C, and 15N nuclei in the subdomain. Of particular significance, complete1H resonance assignments were obtained for all of the leucine, isoleucine, valine, phenylalanine, and alanine side chains (among others) in the subdomain; assignment of NOE cross-peaks derived from these side chains were critical in defining the hydrophobic core. A superposition of a set of structures that are equally consistent with more than 900 NMR-derived constraints is shown in Fig.5. These structures are a fair representation of the full range of structures that are consistent with the NMR data. Structural statistics for residues 2–50 are summarized in Table I. Coordinates for the subdomain have been deposited in the Protein Data Bank (PDB), where it has been assigned accession number 1ND9. The NMR results show that the first 50 amino acids of IF2 form a compact structure consisting of three β-strands and three short α-helices. The three helices are nearly orthogonal, and are located on the same side of an antiparallel twisted sheet formed by three β-strands. Strands β1 (residues 3–6) and β2 (residues 29–32) are linked by helices α1 (residues 8–12) and α2 (residues 16–26), strand β3 (residues 35–39) is connected to β2 by a short loop, and the compact subdomain terminates with helix α3 (residues 42–50). Alignments of IF2 sequences from different species show that hydrophobic residues Ile-6, Leu-9, Val-17, Leu-20, Val-21, Phe-24, Ala-27, Ile-29, Val-37, Leu-45, Ile-46, and Leu-49 are conserved in a wide range of species (Figs. 6 and 7); these residues are all buried and form the core of the subdomain structure. Aribbon diagram depicting the fold of the subdomain is shown in Fig. 5.Figure 7Alignment of the N-terminal 50 amino acids of IF2 in a diverse set of bacteria. The alignment provides evidence that the structure of the 50 amino acid α/β motif at the N terminus of Domain I is well conserved among a wide range of bacterial species. The conserved residues that form the hydrophobic core of the 50 amino acid N-terminal motif are shown in red and indicated withred arrows. Sequences of IF2 were chosen so as to represent a much more divergent set of bacteria than that shown in Fig. 5.E. coli belongs to the γ subdivision of the proteobacteria; Desulfovibrio desulfuricans andGeobacter metallireducens belong to the Δ subdivision of the proteobacteria; Campylobacter jejuni belongs to the ε subdivision of the proteobacteria; B. subtilis, B. stearothermophilus, Streptococcus pneumoniae, andLactococcus lactis are members of the firmicutes;Nostoc punctiforme, Prochlorococcus marinus, Synechocystis, Thermosynechococcus elongatus, and Trichodesmium erythraeum belong to various subdivisions of the cyanobacteria; Deinococcus radiodurans and Thermus thermophilus belong to the thermus/deinococcus group; Thermotoga maritima represents the thermotogae; Chlorobium tepidum represents the chlorobi, and Chlamydia pneumoniae represents the chlamydiae group. Alignment of the conserved hydrophobic residues does not require any gaps except for the single amino acid deletion between residues 35 and 36 in the E. coli sequence; this deletion also occurs in some of the other β and γ proteobacteria (Fig. 6) and is in a loop region of the structure. Although an excellent alignment was found for the first 50 amino acids of IF2 from each species shown, significant similarity was not found for the amino acids in the region corresponding to 51–157 of the E. coli sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In previous work, monoclonal antibodies were generated against IF2 fromE. coli (17Mortensen K.K. Kildsgaard J. Moreno J.M. Steffensen S.A. Egebjerg J. Sperling-Petersen H.U. Biochem. Mol. Biol. Int. 1998; 46: 1027-1041PubMed Google Scholar). One of these antibodies was epitope mapped on native full-length IF2 to the region of β2-β3 and the loop connecting these strands, suggesting that this region may be solvent-exposed in the full-length IF2; however, we note that epitope mapping is not an unambiguous indicator of solvent-exposed residues. The coordinates for the structure of the IF2 N-terminal subdomain were compared against a data base of known structures using the Vector Alignment Search Tool (VAST), located at the NCBI Web page, and the DALI search tools (29Holm L. Sander C. J. Mol. Biol. 1993; 233:" @default.
• W2110685170 created "2016-06-24" @default.
• W2110685170 creator A5015366477 @default.
• W2110685170 creator A5044147191 @default.
• W2110685170 creator A5064902465 @default.
• W2110685170 creator A5072967213 @default.
• W2110685170 date "2003-05-01" @default.
• W2110685170 modified "2023-09-27" @default.
• W2110685170 title "A Conserved Structural Motif at the N Terminus of Bacterial Translation Initiation Factor IF2" @default.
• W2110685170 cites W1188919047 @default.
• W2110685170 cites W1554349566 @default.
• W2110685170 cites W1575999626 @default.
• W2110685170 cites W1586232998 @default.
• W2110685170 cites W1965198018 @default.
• W2110685170 cites W1968366326 @default.
• W2110685170 cites W1970438595 @default.
• W2110685170 cites W1972356897 @default.
• W2110685170 cites W1975459052 @default.
• W2110685170 cites W1987027605 @default.
• W2110685170 cites W1990180127 @default.
• W2110685170 cites W1990595341 @default.
• W2110685170 cites W1995017064 @default.
• W2110685170 cites W1995121880 @default.
• W2110685170 cites W2000483347 @default.
• W2110685170 cites W2001599040 @default.
• W2110685170 cites W2015308335 @default.
• W2110685170 cites W2015642465 @default.
• W2110685170 cites W2019994138 @default.
• W2110685170 cites W2022058405 @default.
• W2110685170 cites W2023856819 @default.
• W2110685170 cites W2024174659 @default.
• W2110685170 cites W2024684906 @default.
• W2110685170 cites W2025121827 @default.
• W2110685170 cites W2030702979 @default.
• W2110685170 cites W2034743409 @default.
• W2110685170 cites W2066896073 @default.
• W2110685170 cites W2069602336 @default.
• W2110685170 cites W2071390057 @default.
• W2110685170 cites W2071804506 @default.
• W2110685170 cites W2076819588 @default.
• W2110685170 cites W2088230790 @default.
• W2110685170 cites W2094509735 @default.
• W2110685170 cites W2098511780 @default.
• W2110685170 cites W2110267171 @default.
• W2110685170 cites W2129879061 @default.
• W2110685170 cites W2158714788 @default.
• W2110685170 cites W2169821755 @default.
• W2110685170 doi "https://doi.org/10.1074/jbc.m212960200" @default.
• W2110685170 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12600987" @default.
• W2110685170 hasPublicationYear "2003" @default.
• W2110685170 type Work @default.
• W2110685170 sameAs 2110685170 @default.
• W2110685170 citedByCount "33" @default.
• W2110685170 countsByYear W21106851702012 @default.
• W2110685170 countsByYear W21106851702013 @default.
• W2110685170 countsByYear W21106851702015 @default.
• W2110685170 countsByYear W21106851702016 @default.
• W2110685170 countsByYear W21106851702018 @default.
• W2110685170 countsByYear W21106851702022 @default.
• W2110685170 crossrefType "journal-article" @default.
• W2110685170 hasAuthorship W2110685170A5015366477 @default.
• W2110685170 hasAuthorship W2110685170A5044147191 @default.
• W2110685170 hasAuthorship W2110685170A5064902465 @default.
• W2110685170 hasAuthorship W2110685170A5072967213 @default.
• W2110685170 hasBestOaLocation W21106851701 @default.
• W2110685170 hasConcept C104317684 @default.
• W2110685170 hasConcept C105580179 @default.
• W2110685170 hasConcept C121332964 @default.
• W2110685170 hasConcept C132677234 @default.
• W2110685170 hasConcept C142587794 @default.
• W2110685170 hasConcept C149364088 @default.
• W2110685170 hasConcept C185592680 @default.
• W2110685170 hasConcept C24890656 @default.
• W2110685170 hasConcept C32276052 @default.
• W2110685170 hasConcept C33277056 @default.
• W2110685170 hasConcept C4718897 @default.
• W2110685170 hasConcept C515207424 @default.
• W2110685170 hasConcept C55493867 @default.
• W2110685170 hasConcept C57711820 @default.
• W2110685170 hasConcept C62493599 @default.
• W2110685170 hasConcept C70721500 @default.
• W2110685170 hasConcept C86803240 @default.
• W2110685170 hasConcept C95444343 @default.
• W2110685170 hasConceptScore W2110685170C104317684 @default.
• W2110685170 hasConceptScore W2110685170C105580179 @default.
• W2110685170 hasConceptScore W2110685170C121332964 @default.
• W2110685170 hasConceptScore W2110685170C132677234 @default.
• W2110685170 hasConceptScore W2110685170C142587794 @default.
• W2110685170 hasConceptScore W2110685170C149364088 @default.
• W2110685170 hasConceptScore W2110685170C185592680 @default.
• W2110685170 hasConceptScore W2110685170C24890656 @default.
• W2110685170 hasConceptScore W2110685170C32276052 @default.
• W2110685170 hasConceptScore W2110685170C33277056 @default.
• W2110685170 hasConceptScore W2110685170C4718897 @default.
• W2110685170 hasConceptScore W2110685170C515207424 @default.
• W2110685170 hasConceptScore W2110685170C55493867 @default.
• W2110685170 hasConceptScore W2110685170C57711820 @default.
• W2110685170 hasConceptScore W2110685170C62493599 @default.

• next