NMR structure of protein yjbR from <i>Escherichia coli</i> reveals ‘double‐wing’ DNA binding motif

126-residue protein yjbR (gi|85674275, SwissProt/TrEMBL ID YJBR_ECOLI, access number P0AF50) encoded by gene YJBR_ECOLI from Escherichia coli has no significant sequence similarity with any protein with known three-dimensional structure. Protein yjbR was selected by the Protein Structure Initiave-2 of the United States National Institutes of Health and was assigned to the Northeast Structural Genomics Consortium (NESG; http://www.nesg.org) (NESG target ID ER226). Protein yjbR belongs to Protein Family (Pfam1) PF04237 which contains 133 proteins. No functional annotations are so far available. Here we report the high-quality NMR solution structure of protein yjbR, which was determined by recently developed rapid NMR data collection and analysis protocol for high-throughput protein structure determination.2 Protein yjbR (YJBR_ECOLI) was cloned, expressed, and purified following standard protocols to produce a uniformly U-13C,15N-labeled protein sample.3 Briefly, the full length ER226 gene from Escherichia coli was cloned into a pET21 (Novagen) derivative, yielding the plasmid pER226-21.1. 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 pER226-21.1 and cultured in MJ9 minimal medium containing (15NH4)2SO4 and U-13C-glucose as sole nitrogen and carbon sources.4U-13C,15N yjbR was purified using an AKTAxpress (GE Healthcare) based two-step protocol consisting of IMAC (HisTrap HP) and gel filtration (HiLoad 26/60 Superdex 75) chromatography. The final yield of purified U-13C, 15N yjbR (>98% homogenous by SDS-PAGE; 14.6 kDa by MALDI-TOF mass spectrometry) was about 79.5 mg/L. The final samples of U-13C, 15N labeled yjbR was prepared at a concentration of ∼1.1 mM in 95% H2O/5% D2O solution containing 20 mM MES, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3 at pH 6.5. An isotropic overall rotational correlation time of ∼8 ns was inferred from 15N spin relaxation times, indicating that the protein is monomeric in solution. This conclusion was further confirmed by gel-filtration and dynamic light scattering. All NMR spectra were recorded at 25°C. Five through-bond correlated G-matrix Fourier transform5 (GFT) NMR experiments4-6 were collected for backbone and side chain resonance assignment on a Varian INOVA 600 spectrometer equipped with a cryogenic probe (total measurement time, 54 h), and a heteronuclear 15N/13Caliphatic/13Caromatic-resolved [1H,1H] NOESY spectrum6 (mixing time, 60 ms) was acquired on a Varian INOVA 750 spectrometer equipped with a conventional probe (measurement time, 24 h) to derive 1H-1H distance constraints. Spectra were processed and analyzed with the programs NMRpipe7 and XEASY,8 respectively. Sequence specific backbone (HN, Hα, N, Cα) and Hβ/Cβ resonance assignments were obtained by using (4,3)D HNNCαβCα/CaαbβCα(CO)NHN and (4,3)D HαβCαβ (CO)NHN experiments. Side-chain assignments were accomplished by using aliphatic and aromatic (4,3)D HCCH.4 Assignments were obtained for 93% of the assignable backbone (excluding the N-terminal NH3+, the Pro 15N, and the 13C′ shifts) and 13Cβ, and for 93% of the side chain chemical shifts (excluding Lys NH3+, Arg NH2, OH, side chain 13C′, and aromatic quaternary 13C shifts; Table I). Stereospecific assignments were obtained for 25% of the β-methylene groups exhibiting nondegenerate proton chemical shifts, and for 37% of the Val and Leu isopropyl moieties (Table I). Chemical shifts were deposited in the BioMagResBank (accession code: 7281). Upper distance limit constraints for structure calculations were extracted from NOESY (Table I). In addition, backbone dihedral angle constraints were derived from chemical shifts as described12 for residues located in well defined β-strands A–E and α-helix I–III (Table I) by using the program TALOS. The program DYANA was used to convert the volume integrals into the upper distance limit constraints (.upl) and TALOS constraints into angle constraints (.aco). The programs CYANA13, 14 and AUTOSTRUCTURE15 were used in parallel to automatically assign long-range NOEs.2 The final structure calculations were performed using version 2.1 of CYANA14 and CNS16 (see http://www.las.jp/prod/cyana/eg). The structure of protein yjbR was determined by a recently developed protocol2 for rapid NMR data collection based on GFT NMR method and simultaneous 3D 15N, 13Caliphatic, 13Caromatic resolved [1H,1H]-NOESY. The statistics of the structure determination are summarized in Table I and show that a high-quality structure was obtained. Protein yjbR contains six β-strands (A–F) comprising residues 17–20, 26–30, 36–41, 46–49, 66–69, and 79–82, respectively (Fig. 1; PDB ID, 2FKI). Those form a single sheet to which four α-helices (I–IV) comprising, respectively, residues 3–11, 52–65, 90–105, and 110–116 are attached on one side of the sheet. β-strands A–F are arranged in antiparallel fashion with topology A(↓), B(↑), C(↓), D(↑), F(↓), and E(↑). α-helix I is attached to β-strands A and B, while the long α-helix III is attached with β-strands B, C and D. The C-terminal α-helix IV is oriented nearly orthogonal to helices I and III and is located close to the loops connecting α-helix I and β-strands A and B, and β-strand B and C. α-helix II is more disordered in solution and in contact with α-helix III. NMR structure of protein yjbR. (a) The 20 CYANA conformers with the lowest residual CYANA target function representing the NMR solution structure of protein yjbR are shown after superposition of the backbone heavy atoms N, Cα, and C′ atoms of the regular secondary structure elements (Table I). (b) Ribbon drawing of the CYANA conformer with the lowest residual target function value (Table I). The α-helices (I–III) are shown in red and yellow, the β-strands (A–F) are in cyan, other polypeptide segments are in grey, and the N- and C-terminal ends of the protein are indicated as ‘N’ and ‘C’. (c) Sausage diagram of 20 superimposed conformers in the standard orientation of (a). For the presentation of the backbone, a spline function was drawn through the Cα positions and the thickness of the cylindrical rod is proportional to the mean of the global displacements of the 20 CYANA conformers calculated after superposition as in described for (a). Figures (a)–(c) were generated using the program MOLMOL.17 A search for structurally similar proteins using the program Dali18 yields five structures with z-score ≥5.0. The X-ray structure (PDB ID 2a1v) of protein q9rrt5 from Deinococcus radiodurans (gi|11612676; SwissProt/TrEMBL ID, Q9RRT5_DEIRA; sequence identity with protein yjbR, 26%; Dali z-score, 11.8) was solved by the New York Genomics Research Consortium and is most similar to protein yjbR (Dali z-score: 11.8). However, no functional annotation is available for this protein. Dali further identifies the structure of protein cyaY (1ew4) from Escherichia coli (85674274; Q9RRT5_DEIRA; 8%; 5.8), a mitochondrial protein involved in iron homeostasis,19 as well as the structures of protein scyt (2bho) from Yersinia enterocolitica (28302111; SCYT_YEREN; 10%; 5.5) and of protein q9k2l2 (1s28) from Pseudomonas syringae (55978466; Q9K2L2_PSESH; 7%; 5.0). The latter two proteins function as chaperones. However, we found that for none of these three proteins, electrostatic properties or key functional residues are conserved in protein yjbR. In contrast, a likely functional annotation for protein yjbR (http://honiglab.cpmc.columbia.edu/nesg/ER226/ER226_web.html; see this website also for results obtained with other programs searching for structurally similar proteins) can be inferred from the structural similarity to the C-terminal domain (1kaf) of the bacteriophage T4 transcription factor MotA (29345244; MOTA_BPT4; 5%; 5.0). This domain, named MotCF, was likewise identified by the program SSM20 as being structurally similar to protein yjbR. MotCF contains a recently discovered novel DNA-binding motif called “double wings”19, 21, 22 (Fig. 2), in which an exposed β-sheet specifically interacts with the minor and major groove of the DNA duplex, respectively, through the loops between strands A and B, and between strands E and F (Fig. 2). The corresponding protein surface is highly positively charged, as is expected for DNA-binding, and possesses solvent exposed aromatic side-chains mediating binding specificity. In spite of the very low sequence identity (5%) of MotCF and protein yjbR, the electrostatic surfaces in the putative DNA binding region are quite similar. Moreover, most of the residues conserved between MotCF and protein yjbR are located in the β-sheet putatively interacting with DNA (Fig. 2). The sequence motif Asn-Lys-Ala-His-Trp(74–78), which is ultra-highly conserved within Pfam PF04237, appears to be the structural analogue of the putative DNA binding residues located in the loop between strands E and F of MotCF (Fig. 2). Thus, Trp 78 in protein yjbR can be considered to play a similar role as Tyr 191 in MotCF which is assumed to interact with the major groove of the DNA duplex (Fig. 2). Similarly, His 21 and Trp 24 in protein yjbR might play the same role as Tyr 134 in MotCF. This residue mediates specific DNA binding in the minor groove. (a) Model of the MotCF-DNA complex.22 Residues which are putatively mediating specific DNA binding (Lys 129, Lys 130, Tyr 134, Arg 135, Lys 144, Arg 145, Arg 150, Arg 161, Phe 163, Tyr 165, Lys 166, Lys 183, Lys 186, Tyr 191, Lys 195) are shown in yellow. (b) Ribbon drawing of the NMR structure of protein yjbR in which conserved residues of the exposed β-sheet are highlighted. The side-chains of residues which are conserved in all 122 members of Pfam PF04237 are shown (His 21, Trp 24, Gln 28, Lys 30, Val 34, Leu 35, Met 38, Arg 45, Als 47, Ser 49, Lys 51, Thr 52, Arg 68, Ser 70, Arg 71, His 72, Leu 73, Asn 74, Lys 75, Ala 76, His 77, Trp 78, and Thr 80). Specifically, the side chains of residues which are also conserved in MotCF (His 21, Trp 24, Lys 30, Lys 51, His 77, Trp 78) are shown in green. Protein yjbR belongs to COG2315. Note that the overall sequence identity between proteins MotCF and yjbR is only 5%. Taken together, we report here the NMR solution structure of protein yjbR from Escherichia coli. Comparison with the X-ray structure of MotCF and considering the location of residues conserved within Pfam04237 strongly suggest that all members of Pfam04237 represent DNA binding domains comprising the ‘double wing’ motif.22

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.