Ensemble of the 20 lowest energy structures out of 100 calculated.
NMR structure of the hypothetical protein encoded by the YjbJ gene from Escherichia coli
Article first published online: 29 APR 2002
Copyright © 2002 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 47, Issue 4, pages 572–574, 1 June 2002
How to Cite
Pineda-Lucena, A., Liao, J., Wu, B., Yee, A., Cort, J. R., Kennedy, M. A., Edwards, Aled. M. and Arrowsmith, C. H. (2002), NMR structure of the hypothetical protein encoded by the YjbJ gene from Escherichia coli. Proteins, 47: 572–574. doi: 10.1002/prot.10120
- Issue published online: 29 APR 2002
- Article first published online: 29 APR 2002
- Manuscript Accepted: 4 JAN 2002
- Manuscript Received: 3 JAN 2002
- Ontario Research and Development Challenge Fund
- National Institutes of Health. Grant Number: P50 GM62413-02
Here we describe the solution structure of YjbJ (gi|418541) as part of a structural proteomics project on the feasibility of the high-throughput generation of samples from Escherichia coli for structural studies. YjbJ is a hypothetical protein from E. coli of unknown function.1 It is conserved, showing significant sequence identity to four predicted prokaryotic proteins, also of unkown function [Fig. 1(A)]. These include gi|16762921 from Salmonella enterica (S. typhi), gi|17938413 from Agrobacterium tumefaciens, gi|16265654 from Sinorizhobium meliloti, and gi|15599932 from Pseudomona aeruginosa. The structure of YjbJ reveals a new variation of a common motif (four-helix bundle) that could not be predicted from the protein sequence. Although the biochemical function is unknown, the existence of patterns of conserved residues on the protein surface suggest that the fold and function of all these proteins could be similar.
Materials and Methods.
A recombinant protein consisting of the full sequence of YjbJ (69 amino acids) was expressed in E. coli BL21-DE3 cells containing the pET-15b expression vector (Novagen). Cells were grown at 37°C to an OD600 of 0.6 and induced with 1 mM IPTG for 5 h at 25°C. The protein was purified to homogeneity by using metal affinity chromatography. Subsequently, the N-terminal tag was removed by using thrombin and benzamidine-sepharose. The purified protein contained the complete sequence of YjbJ plus three additional N-terminal residues (Gly-Ser-His) remaining after proteolytic cleavage of the His6 affinity tag. U-15N and U-13C,15N samples were produced in standard M9 media supplemented with 15N ammonium chloride (1 g/L) and 13C glucose (2 g/L). 15N-labeled or 13C/15N-labeled protein solution was prepared in 25 mM sodium phosphate (pH = 6.5), 150 mM NaCl, 1 mM DTT, 95% H20/5% D2O. The concentration of the purified protein ranged between 1.0 and 1.5 mM.
All NMR spectra were recorded at 25°C on a Varian INOVA 600-MHz spectrometer equipped with pulsed field gradient triple-resonance probes. Linear prediction was used in the 13C and 15N dimensions to improve the digital resolution. Spectra were processed by using the NMRPipe software package2 and analyzed with XEASY.3 SPSCAN4 was used to convert nmrPipe formatted spectra into XEASY. The assignments of the 1H, 15N, and 13C resonances were based on the following experiments: CBCA(CO)NH, HNCACB, CC(CO)NH-TOCSY, HNHA, HC(CO)NH-TOCSY, and HCCH-TOCSY.5, 6 The backbone resonance assignment was achieved mainly by the combined analysis of the HNCACB and CBCA(CO)NH data. The side-chain resonances were identified mainly by the analysis of HCCH-TOCSY. Aromatic ring resonances were assigned on the basis of the analysis of heteronuclear NOESY. In the 1H-15N HSQC, 99% backbone amide resonances were assigned. Of the other backbone resonances, 99% have been assigned for Cα, and 99% for Hα. Moreover, 97% aliphatic side-chains have been assigned for YjbJ.
For structure calculation purposes, a simultaneous 15N- and 13C-NOESY-HSQC7 (τm = 150 ms) was acquired. NOE cross-peak assignment was obtained by using a combination of manual and automatic procedures. An initial fold of the protein was calculated on the basis of unambiguously assigned NOEs, with subsequent refinement using the NOAH module in the program DYANA.8 Peak analysis of the NOESY spectra were generated by interactive peak picking with the program XEASY. Backbone dihedral restraints were derived from 1Hα and 13Cα secondary chemical shifts using TALOS.9 The program MOLMOL10 was used to analyze the energy-minimized conformers and to prepare pictures of the structures.
Results and Discussion.
YjbJ adopts a four-helix bundle structure [Fig. 1(B)] with residues in all four helices as well as in the turn regions defining a compact structural domain. Helix α1 extends from residue Trp10 to Gly22, whereas the α2, α3, and α4 helices span residues Thr25-Glu33, Arg36-Arg46, and Lys51-Arg64, respectively.
The three-dimensional structure of YjbJ was determined by using a torsion angle dynamics protocol from a total of 2036 NMR-derived constraints. A superposition of 20 low-energy structures is shown in Figure 2, and the structural statistics are given in Table I. The results obtained for the ordered regions of the protein are virtually identical. This is probably due to the small size of YjbJ and the short length of the loops connecting the ordered regions of the protein. A 3D structure search using DALI11 showed that YjbJ shares some structural homology to the α-helical regions of the Bchi subunit of magnesium chelatase and T7 DNA polymerse (PDB accession numbers 1g8p and 1t7p, respectively). In both cases, the similarity is based on the existence of four sequential α-helical elements in these proteins, but the spatial orientation and length of these α-helices are very different compared to YjbJ.
|Sequential (|i-j| = 1)||423|
|Medium range (2 ≤ |i-j| ≤ 4)||628|
|Long range (|i-j| > 4)||463|
|Hydrogen bonds||31 × 2|
|Dihedral angle restraints|
|Backbone atoms||0.23 ± 0.10|
|All heavy atoms||0.99 ± 0.21|
|Backbone atoms||0.23 ± 0.10|
|All heavy atoms||0.95 ± 0.18|
|Residues in most favored regions (%)||86|
|Residues in additional allowed regions (%)||14|
|Residues in generously allowed regions (%)||0|
|Residues in disallowed regions (%)||0|
The chemical shifts have been submitted to the BMRB (accession # 5105), and the structure ensemble has been submitted to the PDB (accession # 1JYG).
The authors thank A. Semesi for technical assistance. All the spectra were performed at the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the U.S. DOE Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory, operated by Batelle for the DOE. AME and CHA are Scientists of the Canadian Institutes of Health Research.
- 7Simultaneous acquisition of N15-edited and C13-edited NOE spectra of proteins dissolved in H2O. J Magn Reson 1994; B103: 197–201., , , , .