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NMR structure of the hypothetical protein AQ‐1857 encoded by the Y157 gene from Aquifex aeolicus reveals a novel protein fold

Proteins Structure Function and Bioinformatics2004Vol. 54(4), pp. 794–796

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Duanxiang Xu, Gaohua Liu, Rong Xiao, T.B. Acton, Sharon Goldsmith‐Fischman, Barry Honig, G.T. Montelione, Thomas Szyperski

Abstract

Uniformly (U) 13C, 15N-labeled AQ-1857 was cloned, expressed and purified following standard protocols. Briefly, the full length gene (Y157_AQUAE) from Aquifex aeolicus was cloned into a pET21d (Novagen) derivative, yielding the plasmid pQR6-21. 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 pQR6-21, and cultured in MJ minimal medium containing (15NH4)2SO4 and U-13C-glucose as sole nitrogen and carbon sources. U-13C,15N AQ-1857 was purified 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 AQ-1857 (> 97% homogeneous by SDS-PAGE; 14.4 kDa by MALDI-TOF mass spectrometry) was about 10 mg/L. In addition, a sample which was U-15N and 5% biosynthetically directed fractionally 13C-labeled was generated for stereospecific assignment of isopropyl methyl groups.8 Two samples of 5%13C,U-15N and U-13C,15N AQ-1857 were prepared at concentrations of 1.0 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. All NMR data were collected at 20°C on Varian INOVA 600 and 750 spectrometers. The spectra were processed and analyzed using the programs NMRPipe9 and XEASY,10 respectively. Resonance assignments were obtained as described11 using a suite of reduced-dimensionality NMR experiments, including 3D HNNCAHA, HαβCαβ(CO)NHN, HCCH-COSY, and 2D HBCB(CGCD)HD. These data were complemented by conventional12 HNNCACB and HC(C)H TOCSY experiments. Assignments were obtained for 93% of the backbone and 13Cβ, and for 91% of the side chain chemical shifts. Stereospecific assignments were obtained for 44% of the β-methylene groups exhibiting non-degenerate proton chemical shifts, and for all Val and Leu isopropyl moieties. The chemical shifts were deposited in the BioMagResBank (accession code: 5683). Upper distance limit constraints for structure calculations were obtained from 3D 15N-and 13C-resolved [1H,1H]-NOESY12 (Table I). In addition, 3JHNα scalar couplings measured in 3D HNNHA12 yielded ϕ-angle constraints, and backbone dihedral angle constraints were derived from chemical shifts as described13 for residues located in regular secondary structure elements (Table I). Structure calculations were performed using the program DYANA.14 Statistics for the structure determination (Table I) show that a high-quality NMR structure was obtained (Fig. 1). AQ-1857 (PDB ID: 1NWB) contains seven β-strands A to F and two α-helices. A(↓), F(↓) and G(↑) form a 3-stranded, and D(↓), E(↑), B(↑) and C(↓) form a 4-stranded sheet. The two sheets form a “sandwich” being rotated by ∼45 degrees relative to each other (Fig. 2). The segment 40–45 and the C-terminal tail 102–116 are flexibly disordered in solution. The 20 DYANA conformers with the lowest residual DYANA target function chosen to represent the NMR solution structure of AQ-1857 are shown after superposition of the backbone heavy atoms N, Cα and C′ of the regular secondary structure elements for minimal RMSD. A: The novel fold of AQ-1857: ribbon drawing of the DYANA conformer with the lowest residual target function value. The α-helices I and II are shown in red and yellow, the β-strands A to G 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’. B: Same as in (A), but rotated by 90° about the vertical axis. α-Helix I: residues 14–25, II: 77–79; β-Strand A: 10–12, B: 33–36, C: 52–53, D: 63–65, E: 69–72, F: 84–89, G: 94–99. The NMR structure of AQ-1857 is the first structure representative of the larger HesB family2, 3 of proteins. No meaningful structural homologues were identified using the programs SKAN,15 DALI,16 or CE.17 This finding strongly supports the notion that AQ-1857 possesses a hitherto uncharacterized, novel fold (Fig. 2). Interestingly, the PROSITE consensus pattern for the HesB family spans the flexibly disordered C-terminal tail of the protein. In fact, two of the three cysteinyl residues which have been proposed to be involved in iron-sulfur cluster assembly in members of this family5, 6 are located in this tail (not shown in Fig. 1; the third cysteine is located in position 43 in the loop connecting β-strands 2 and 3). It is thus very likely that the flexibly disordered tail is of functional importance, and it is tempting to speculate that this tail adopts an ordered conformation only upon involvement in Fe-S cluster assembly. This work was supported by the National Institutes of Health (P50 GM62413-01), the National Science Foundation (MCB 00075773 to T.S.; DBI-9904841 to B.H), and the Center for Computational Research at UB.

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Abstract

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