NMR structure of protein yqbG from <i>Bacillus subtilis</i> reveals a novel α‐helical protein fold

131-residue protein yqbG (gi|16079665, SwissProt/TrEMBL ID YQBG-BACSU, access number P45923) encoded by gene YQBG from Bacillus subtilis has no significant sequence similarity with any protein with known three-dimensional structure and thus was selected as a target (ID APC1551) by the Midwest Center of Structural Genomics (MCSG; http://www.mcsg.anl.gov) for X-ray crystal structure determination. Because protein yqbG is highly soluble and monodispersed while no diffraction quality crystals could be obtained, it was transferred to NMR division of the Northeast Structural Genomics consortium (NESG; http://www.nesg.org) for structure determination (NESG target ID SR215). Notably, yqbG belongs to temporarily created Protein Family (Pfam1) PB071099, which contains only protein lin1273 (45% sequence identity with yqbG) as a second member. An iterative PSI-Blast2 search of the nonredundant Genbank reveals that yqbG exhibits significant sequence identity to a third protein, yqbG (gi|52785100) from Bacillus licheniformis (55% identity). All three proteins belong to PIRSF family SF007500.3 So far, no functional annotations are available. Here we report the high-quality NMR solution structure of protein yqbG,, which exhibits a novel α-helical fold. Uniformly (U) 13C,15N-labeled yqbG was cloned, expressed, and purified by following standard protocols. Briefly, the full-length gene from Bacillus subtilis was cloned into a pET21 (Novagen) derivative, yielding the plasmid pSR215-21.3. The resulting construct contains eight nonnative residues at the C-terminus (LEHHHHHH) that facilitate protein purification. Escherichia coli BL21 (DE3) pMGK cells, a rare codon enhanced strain, were transformed with pSRF215-21.3 and cultured in MJ9 minimal medium containing (15NH4)2SO4 and U-13C-glucose as sole nitrogen and carbon sources.4 U-13C,15N yqbG was purified by using a two-step protocol consisting of Ni-NTA affinity (Qiagen) and gel filtration (HiLoad 26/60 Superdex 75; Amersham Biosciences) chromatography. The final yield of purified U-13C,15N yqbG (>98% homogenous by SDS-PAGE; 16.6 kDa by MALDI-TOF mass spectrometry) was about 13.5 mg/L. The final samples of U-13C,15N labeled yqbG were prepared at a concentration of about 0.9 mM in 95% H2O/5% D2O solution containing 20 mM MES, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, and 0.02% NaN3 at pH 6.5. All NMR spectra were recorded at 25°C. Five G-matrix Fourier transform5 (GFT) NMR experiments5-7 were performed for resonance assignment on a Varian INOVA 600 spectrometer equipped with a cryogenic probe (total measurement time: 104 h), and a simultaneous 15N/13Caliphatic/13Caromatic-resolved NOESY spectrum7 was acquired on a Varian INOVA 750 spectrometer equipped with a conventional probe (measurement time: 23 h) to determine 1H–1H distance constraints. Spectra were processed and analyzed with the programs PROSA8 and XEASY9, respectively. Sequence specific backbone (HN, Hα, N, Cα) and Hβ/Cβ resonance assignments were obtained by using (4,3)D HNNCαβCα/CαβCα(CO)NHN6 and (4,3)D HαβCαβ(CO)NHN. Side-chain spin system identification was accomplished by using aliphatic and aromatic (4,3)D HCCH.7 Assignments were obtained for 100% of the assignable backbone (excluding the N-terminal NH, the Pro 15N and the 13C′ shifts) and 13Cβ, and for 99% of the side-chain chemical shifts (excluding Lys NH, Arg NH2, OH, side-chain 13C′, and aromatic quaternary 13C shifts; Table I). Stereospecific assignments were obtained for 55% of the β-methylene groups exhibiting nondegenerated proton chemical shifts and for 80% of the Val and Leu isopropyl moieties (Table I). Chemical shifts were deposited in the BioMagResBank (accession code: 6366). Upper distance limit constraints for structure calculations were obtained from NOESY (Table I). In addition, backbone dihedral angle constraints were derived from chemical shifts as described10 for residues located in globally well-defined helices I–V (Table I). The programs CYANA11, 12 and AUTOSTRUCTURE13 were used in parallel to automatically assign long-range NOEs. Coinciding assignments (“consensus assignments”) were retained and established the starting point for manual completion of iterative NOE assignment, peak picking, and structure calculation. The final structure calculations were performed by using version 2.0 of CYANA,12 (see: http://www.las.jp/prod/cyana/eg). NMR structure of protein yqbG revealing a novel α-helical fold. a: 20 CYANA conformers with the lowest residual target function chosen to represent the NMR solution structure of protein yqbG are shown after superposition for minimal root-mean-square deviation (RMSD) of the backbone heavy atoms N, Cα, and C′ of globally well-defined α-helices I–V (Table I). b: Ribbon drawing of the CYANA conformer with the lowest target function. The α-helices are shown in red and yellow, residue numbers (helix I: residues 6–12, II: 16–20, III: 23–41, IV: 55–72, V: 105–111, 125–129) are indicated, and the N- and C-termini of the polypeptide chain are labeled with N and C. c: Sausage presentation of backbone and best defined side-chains. A spline function was drawn through the Cα positions of residues 3–113, where the thickness of the cylindrical rod is proportional to the mean of the global displacements of the Cα atoms in the 20 CYANA conformers calculated after superposition as described in (a). The α-helices I–V are shown in red, and coil regions are displayed in gray. A superposition of the best defined side-chains with the lowest global displacement for the side-chain heavy atoms (for residue numbers, see Table I) is shown in blue to indicate precision of the determination of side-chain conformations. The statistics of the yqbG structure determination (Table I) show that a high-quality NMR structure was obtained (Fig. 1). Protein yqbG (PDB ID: 1ZTS) contains five α-helices I–V [Fig. 1(b)] comprising residues 6–12, 16–20, 23–41, 55–72, and 105–111. These five helices are locally and globally well defined. In contrast, the polypeptide segment 74–99 and the C-terminal tail of residues 114–131, which comprise the locally well-defined short α-helix VI (residues 125–129), are disordered in solution (Fig. 1). We speculate that the presence of these long flexible segments prevents crystallization of yqbG. The CATH/GRATH14 structure classification scheme assigns the fold of yqbG to the class “mainly α-helical” having the architecture of an “orthogonal” bundle. No protein with meaningful structural similarity could be identified by using the programs DALI15 or CE.16 The highest z scores are at about the threshold of being significant (i.e., 3.0 and 4.1 for DALI and CE, respectively), but visual inspection reveals that this is due to the presence of the two long helices, helix III and IV [Fig. 1(b)], arranged in an antiparallel manner. Such arrangement of two helices is quite common and does, on its own, not point at significant structural similarity of two protein architectures. Furthermore, except for one protein, different candidates for structural similarity were identified by the two programs. Thus, we conclude that protein yqbG possesses a hitherto uncharacterized α-helical fold [Fig. 1(b)]. Notably, a PROSITE17 motif search indicates that yqbG and its three sequence homologues share the Casein kinase II phosphorylation site Thr-Pro-Asp-Glu/Asp at the N-terminal end (residues 5–8 in yqbG). The authors thank M. Ciaro and K. Cunningham for technical assistance. This work was supported by the National Institutes of Health and the National Science Foundation (T.S.).

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