Unstructured N- and C-termini (residues 1–12, 90–94) excluded
NMR structure of conserved eukaryotic protein ZK652.3 from C. elegans: A ubiquitin-like fold
Article first published online: 30 JUL 2002
Copyright © 2002 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 48, Issue 4, pages 733–736, 1 September 2002
How to Cite
Cort, J. R., Chiang, Y., Zheng, D., Montelione, G. T. and Kennedy, M. A. (2002), NMR structure of conserved eukaryotic protein ZK652.3 from C. elegans: A ubiquitin-like fold. Proteins, 48: 733–736. doi: 10.1002/prot.10197
- Issue published online: 30 JUL 2002
- Article first published online: 30 JUL 2002
- Manuscript Accepted: 2 MAY 2002
- Manuscript Received: 21 MAR 2002
- National Institutes of Health, Protein Structure Initiative. Grant Number: P50 GM62413
Structural proteomics aims to provide one or more representative three-dimensional (3D) structures for every structural domain family in nature. As part of an international effort in structural proteomics, the Northeast Structural Genomics Consortium has targeted clusters of strongly conserved eukaryotic protein families for structural and functional analysis. On this basis, protein ZK652.3 (nesg WR41/WP:CE00949/YOY3_CAEEL/Swiss-Prot P34661/gi|17557033) from Caenorhabditis elegans was selected for structure determination. Sequencing of cDNA libraries shows that homologues of ZK652.3 occur widely in vertebrates and plants (Fig. 1). However, ZK652.3 homologues are conspicuously absent from the yeast and Drosophila genomes. Expression of the ZK652.3 gene has been observed in a transcriptional profile of C. elegans genes, where it was one of a cluster of 89 genes whose expression levels covaried during development.1 The biochemical function of this protein is presently unknown. Here we describe the 3D structure of ZK652.3 determined by nuclear magnetic resonance (NMR) spectroscopy and discuss structural similarities with other proteins that provide clues to potential biochemical functions.
Materials and Methods.
The gene coding for the ZK652.3 protein was subcloned from cDNA clone YK452c8 into expression vector pET15b with a hexa-His N-terminal purification tag, generating plasmid pET15b-WR41. The resulting construct was verified by DNA sequence analysis. E. coli strain BL21(DE3) cell cultures transformed with pET15b-WR41 were grown at 37°C in MJ minimal medium.2 Details of the production and purification of ZK652.3 will be described in detail elsewhere. Sample purity (>95%) and molecular weight (11.5 kDa with purification tag) were verified by SDS-PAGE and MALDI-TOF mass spectrometry. Uniformly 13C, 15N-enriched or 10% 13C, 100% 15N-enriched ZK652.3 protein samples were prepared in 5-mm Shigemi susceptibility-matched NMR tubes, at 1.5 mM protein concentration in H2O solution containing 5% D2O, 10 mM ammonium acetate, 50 mM sodium chloride, and 5 mM DTT at pH 5.50 ± 0.05.
NMR spectra of ZK652.3 were collected at 25°C on 600, 750, and 800 MHz Varian Inova spectrometers at the Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington. Spectra were referenced to external DSS. Two-dimensional 1H-15N HSQC3 and 3D HNHA4 and 15N-edited NOESY-HSQC3 experiments were conducted on [U-15N]-ZK652.3. Two-dimensional 1H-13C HSQC3 and 3D HNCACB,5 HNCO,5 CBCA(CO)NNH,5 CBCACOCAHA,6 HCCH-TOCSY,6 HCC-TOCSY-NNH,7, 8 CCC-TOCSY-NNH,7, 9, 10 CN-NOESY-HSQC,11 and 4D CC-NOESY12 experiments were recorded on [U-13C, U-15N]-ZK652.3. The sample was lyophilized and redissolved in D2O before acquisition of the 4D CC-NOESY data set. NOESY experiments used mixing times of 120 msec (4D CC-NOESY and 3D CN-NOESY-HSQC) or 150 msec (15N-edited NOESY-HSQC).
Spectra were processed and analyzed with Felix (MSI). No chemical shifts for residues L26 and P27 were obtained, and only partial assignments for T21 and F28 were obtained. Stereospecific valine and leucine methyl group assignments were obtained from a 1H-13C-HSQC spectrum recorded on a sample labeled with 10%-13C (Ref. 13). Peak intensities in the NOESY spectra were converted into short (1.8–2.5 Å), medium (1.8–3.0 Å), long (1.8–4.0 Å), and extra-long (1.8–5.0 Å) distance restraints. Pseudoatom corrections for stereochemically ambiguous protons were added to the upper bounds: 1.0 Å for methylene protons, 2.0 Å for chemically equivalent aromatic protons, and 2.4 Å for pairs of methyl groups in leucine and valine residues. All long-range NOEs (i-j > 4) were put initially into the extra-long category, as were all NOEs between side-chains. During structure refinement, the upper bounds of exceptionally intense long-range NOEs were reduced by 0.5–1.0 Å in some cases. Dihedral angle restraints for ϕ were derived from the HNHA experiment.4 Dihedral angle restraints for ψ were added during the later stages of refinement for residues in helix and strand regions where chemical shift and αiNi and αiNi+1 NOESY peak intensities indicated they were appropriate. Hydrogen bond restraints for slowly exchanging amide protons were added late in the refinement when the acceptor atom was identifiable from a preliminary structural ensemble.
A total of 476 NOE distance restraints, 50 hydrogen bond restraints (2 per H-bond), and 71 dihedral restraints (42 ϕ, 29 ψ) were used for calculation of the structural ensemble. Structures were calculated by using X-PLOR 3.840.14 The routines dg_full_embed.inp, dgsa.inp, and refine_gentle.inp were used to generate 26 structures from an extended starting structure. Two high-energy structures were removed to yield an ensemble of 24 structures (Fig. 2). Statistics for this ensemble are compiled in Table I. The structural ensemble and restraints have been deposited in the Protein Data Bank (PDB id 1L7Y), and the chemical shifts have been deposited in BioMagResBank (BMRB-5329).
|Medium-range (1 < |i − j| < 5)||86|
|Long-range (|i − j| ≥ 5)||219|
|Hydrogen bond restraints (2 per H-bond)||50|
|Total number of restraints (all) per residuea||7.8|
|Distance restraint violations|
|Mean number of violations > 0.0 Å||21.1 ± 2.1|
|Maximum number of violations||26|
|Maximum violation (Å)||0.12|
|Mean RMS violation (Å)||0.009 ± 0.001|
|Dihedral restraint violations|
|Mean number of violations > 0.0°||2.6 ± 0.9|
|Maximum number of violations||4|
|Maximum violation (°)||1.5|
|Mean RMS violation (°)||0.12 ± 0.04|
|Mean rms deviation from the average coordinates (Å)|
|Residues 13–21 and 28–89|
|Backbone atoms (Cα, C′, N, O)||0.42 ± 0.09|
|All heavy atoms||0.95 ± 0.10|
|Backbone atoms (Cα, C′, N, O)||0.75 ± 0.25|
|All heavy atoms||1.28 ± 0.25|
|Ramachandran plot (residues 13–89)b|
|In most favored region (%)||87|
|In additional allowed region (%)||10|
|In generously allowed region (%)||2|
|In disallowed region (%)||1|
Results and Discussion.
ZK652.3 adopts a ubiquitin-like α + β fold (or β-grasp fold) with secondary structure elements ordered β- β-α- β- β-α- β along the sequence (Fig. 3). The strands of the β-sheet are ordered 2-1-5-3-4 with strands 2 and 3 antiparallel to the others. Residues 1–12 and 90–94 are not structured, displaying random coil-like backbone chemical shifts and no medium (other than sequential) or long range NOEs. Residues 22–29 appear to be more mobile than the rest of the structured portion of the protein. The backbone amide proton-nitrogen cross peaks for two residues in this region, L26 and F28, could not be located in HSQC spectra, and other residues in the span display strong cross peaks to water and weak cross peaks to other protons in the 15N-edited NOESY spectrum. Consequently, this portion of the structure in not converged in the structural ensemble.
In an initial effort to gain clues to the biochemical function of ZK652.3, its structure was compared with previously determined structures in the Protein Data Bank using Dali.15 The most similar structures identified by Dali were Ras binding domains from RalGDS (PDB id 1LFDchain A, Z = 8.8) and Rap-1a (1C1Ychain B, Z = 8.5), the ubiquitin-like module UBX domain (1H8C, Z = 8.5), and ubiquitin (1UBI, Z = 8.1). All have between 12 and 16% sequence identity with the structured portion of ZK652.3. Two of these structures, 1LFD chain A and 1UBI, are displayed next to ZK652.3 in Figure 3. The Ras binding domains invariably are parts of much larger protein molecules, and although the existence of an isolated Ras binding domain is not inconceivable, attention was focused on ubiquitin and ubiquitin-like modifier proteins. Like ZK652.3, these are small, single-domain proteins.
Several surface features common to ubiquitin and ZK652.3 are notable. Sequence similarity is particularly high in the C-terminus (Fig. 1), although the Gly-Gly-COO− motif with which ubiquitin and many ubiquitin-like modifier proteins terminate is replaced with Val-Gly-His-COO− in ZK652.3, and the chain extends even farther in homologues from other species. Arginine residues R72 and R74 near the C-terminus of ubiquitin, which are essential for its function,16 are conserved in ZK652.3. The side-chains of two residues, I44 and V70, in a critical surface hydrophobic patch on ubiquitin17 are structurally equivalent to I60 and I87 in ZK652.3, whose side-chains also are surface exposed. In the structure of the complex formed by MoaD and MoeB,18 thought to be bacterial progenitors of ubiquitin and ubiquitin E1 activating enzyme, respectively, hydrophobic MoaD residues L59 and F75 at the interface of the two proteins are structurally equivalent to V65 and I87 in ZK652.3. The activating enzyme (E1) in ubiquitin-like modifier systems activates the C-terminal carboxylate of a specific ubiquitin-like modifier protein for subsequent transfer to its conjugating enzyme (E2) then ligation to a target protein by a ligase enzyme (E3).19 We speculate that ZK652.3 may interact with an as yet unidentified activating enzyme in a new ubiquitin-like modification system.
We thank Prof. Y. Kohara for kindly providing cDNA clone YK452c8. We thank J. Aramini, A. Bhattacharya, and K. Gunsalus for helpful discussions, and Ms. D. Magapal for technical assistance. Acquisition and processing of NMR spectra and structure calculations were performed in the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the U.S. Department of Energy Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory and operated for DOE by Battelle (contract KP130103). ZK652.3 from C. elegans is target WR41 of the Northeast Structural Genomics Consortium.
- 1Genomic analysis of gene expression in C. elegans. Science 2000; 290: 809–812., , , , .
- 2High level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli containing (15NH4)2SO4 and 13C6-glucose as sole nitrogen and carbon sources. J Biomol NMR 1996; 7: 131–141., , , , , .
- 3Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques. J Biomol NMR 1994; 4: 845–858., , , .
- 4Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constants in 15N-enriched proteins. J Am Chem Soc 1993; 115: 7772–7777., .
- 5Gradient-enhanced triple-resonance three-dimensional NMR experiments with improved sensitivity. J Magn Reson B 1994; 103: 203–216., .
- 6Pulsed-field gradient-enhanced three-dimensional NMR experiment for correlating 13Cα/β, 13C′, and 1Hα chemical shift in uniformly 13C-labeled proteins dissolved in H2O. J Am Chem Soc 1993; 115: 2055–2057..
- 7An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically enriched proteins. J Am Chem Soc 1992; 114: 10974–10975., , , .
- 8An HCCNH triple-resonance experiment using carbon-13 isotropic mixing for correlating backbone amide and side-chain aliphatic resonances in isotopically enriched proteins. J Magn Reson B 1993; 101: 206–209., .
- 9Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization. J Magn Reson B 1993; 101: 114–119., , .
- 10A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments. J Biomol NMR 1993; 3: 225–231., , , .
- 11Simultaneous acquisition of 15N- and 13C-edited NOE spectra of proteins dissolved in H2O. J Magn Res B 1994; 103: 197–201., , , , .
- 12Increased resolution and improved spectral quality in 4-dimensional 13C/13C-separated HMQC-NOESY-HMQC spectra using pulsed field gradients. J Magn Reson B 1993; 101: 210–213., , , , , , .
- 13Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 1989; 28: 7510–7516., , , , .
- 14X-PLOR Version 3.1. A system for X-ray crystallography and NMR. New Haven, CT: Yale University Press; 1992..
- 15Touring protein fold space with Dali/FSSP. Nucleic Acids Res 1998; 26: 316–319., .
- 16Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin. J Biol Chem 1987; 262: 14213–14221., , , , , , , , , , .
- 17Distinct functional surface regions on ubiquitin. J Biol Chem 2001; 276: 30483–30489., , , .
- 18Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. Nature 2001; 414: 325–329., , , .
- 19All in the ubiquitin family. Science 2000; 289: 563–564..
- 20AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 1996; 8: 477–486., , , , .
- 21MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 1991; 24: 946–950..