NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis Reveals a distinct 5‐helix bundle (2004)

The 82-residue hypothetical protein NMA1147 (SwissProt/TrEMBL ID Q9JR91) encoded in the genome of Neisseria meningitidis is a member of a target sequence cluster1 of the Northeast Structural Genomics Consortium (NESGC; http://www.nesg.org; NESGC cluster ID 15365) that comprises both eukaryotic and bacterial members. The high-quality NMR solution structure of NMA1147 (NESG target ID MR19) reveals a complex bundle of five α-helices, which is composed of an “up-down” 3-helix and an “orthogonal” 2-helix bundle. NMA1147 belongs to the TPR_div1 Pfam2 family (Pf03937; DUF339). This family represents a subfamily of the TPR3 (tetratricopetide repeat) family and exhibits a divergent TPR. The TPR represents an ancient and highly conserved sequence motif spanning two antiparallel helices in proteins involved in various protein-protein interactions.3 The presence of a variation of this motif in NMA1147 suggests that NMA1147 may be involved in distinct protein-protein interactions, and the location of conserved surface residues identifies a putative interaction surface. Uniformly (U) 13C,15N-labeled NMA1147 was cloned, expressed and purified following standard protocols. Briefly, the full length gene (NMA1147) from Neisseria meningitidis was cloned into a pET21 (Novagen) derivative, yielding the plasmid MR19-21. 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 MR19-21, and cultured in MJ minimal medium containing (15NH4)2SO4 and U-13C-glucose as sole nitrogen and carbon sources. U-13C,15N NMA1147 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 NMA1147 (> 97% homogeneous by SDS-PAGE; 11.4 kDa by MALDI-TOF mass spectrometry) was about 50 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.4 Two samples of 5% 13C,U-15N and U-13C,15N NMA1147 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. NMR data were collected at 25°C on Varian INOVA 600 and 750 spectrometers, and spectra were processed and analyzed using the programs NMRPipe5 and XEASY.6 Resonance assignments were obtained as described7 using a suite of reduced-dimensionality (RD) NMR experiments, including 3D HACA(CO)NHN, HNNCAHA, HαβCαβ(CO)NHN, and 2D HBCB(CGCD)HD and 1H-TOCSY relayed HCH-COSY. These data were complemented with conventional8 HNNCACB and HC(C)H TOCSY experiments. Assignments were obtained for 98% of the backbone and 13C,β and for 96% of the side chain chemical shifts. Stereospecific assignments were obtained for 18% of the β-methylene groups exhibiting non-degenerate proton chemical shifts, as well as for all Val and Leu isopropyl moieties. The chemical shifts were deposited in the BioMagResBank (Accession code: 5846). Upper distance limit constraints for structure calculations were obtained from 3D 15N- and 13C-resolved [1H,1H]-NOESY8 (Table I). In addition, 3JHNα scalar couplings measured in 3D HNNHA8 yielded ϕ-angle constraints, and backbone dihedral angle constraints were derived from chemical shifts as described9 for α-helical residues (Table I). Structure calculations were performed using the program DYANA.10 The statistical parameters summarized in Table I are indicative of a high-quality NMR structure [Fig. 1(a)]. NMA1147 (PDB ID: 1PUZ) is composed of five α-helices I to V [Fig. 1(b)]. The program CATH11 assigns the NMA1147 structure to the class “mainly α-helical” and to the architectures “up-down” or “orthogonal” bundle. In fact, helices I, II, and III form an up-down 3-helix bundle, while helices IV and V are arranged in an orthogonal manner. The juxtaposition of these two bundles yields a complex 5-helix bundle [Fig. 1(b)]. Residues conserved among NMA1147 homologues [Fig. 1(c)] are located either in the molecular core, or form a surface cluster involving the last two turns of helix I (Lys 11, Phe 14, Gln 15, Arg 17), the segment connecting helices I and II (Arg 18 and Gly 19), and the N-terminal ends of helices II (Leu 21, Glu 22, Asp 24) and IV (Glu 56). Remarkably, Asp 54, which is neither surface-accessible nor forming a salt bridge in the molecular core, is likewise conserved. Electrostatic surface calculations reveal the presence of an acidic pocket in the region of the conserved Glu 22 at the center of a larger hydrophobic patch (Fig. 2). This pocket is also proximal to conserved surface residues, suggesting that this site is of functional importance. a: The 20 DYANA conformers with the lowest residual DYANA target function chosen to represent the NMR solution structure of NMA1147 are shown after superposition of the backbone heavy atoms N, Cα and C′ of the five α-helices I to V for minimal RMSD Helices I and II form an “up-down” bundle, while helices III to IV are arranged in an “orthogonal” manner. b: Ribbon drawing of the DYANA conformer with the lowest residual target function value. The α-helices are shown in red and yellow, other polypeptide segments are in grey, and the N- and C-terminal ends of the protein are indicated as “N” and “C.” c: Molecule core of the backbone of NMA1147 in the standard orientation of (a). For the presentation of the backbone a spline function was draw through the Cα positions; the thickness of the cylindrical rod is proportional to the mean of the global displacements of the 20 DYANA conformers calculated after superposition as in described for (a). The helices are shown in red, other polypeptide segments are displayed in grey, the conserved side chains of the molecular core are shown in yellow, and the side chains of conserved surface residues are depicted in blue. For clarity, the backbone of the first three residues, which are flexibly disordered in solution [Fig. 1(a)], is not shown. a: Ribbon drawing of NMA1147 [see Fig. 1(b)] in an orientation in which the conserved residue Glu 22 (side chain depicted in magenta) points toward the observer. b: Electrostatic surface potential of NMR1147 in the orientation of (a), demonstrating that a negatively charged pocket is located at about the center of a larger hydrophobic patch. Positive and negative charge densities are colored red and blue, respectively. NMA1147 belongs to the TPR_div1 family (Pf03937; DUF339), a subfamily containing TPR3-like motifs. The consensus sequence motif of the TPR3 repeat, X3-[W/L/Y]-X2-[L/I/M]-[G/A/S]-X2-[Y/L/F]-X8-[A/S/E]-X3-[P/Y/L]-X2-[A/S/L]-X4-[P/K/E], is divergent in NMA1147. The TPR-like sequence spans helices II and III (residues 21–53), in which, however, Phe 27 and Phe 46 are located at positions of relatively small amino acids ([G/A/S] and [A/S/L], respectively) present in the TPR motif. Moreover, the packing of the consensus residues in NMA1147 differs from that found in the TPR motif. Notably, TPR motifs are often occurring as multiple repeats3 whereas NMA1147 contains only a single motif. The programs SKAN,12 DALI,13 and CE14 identify structural homologues which are classified by the program SCOP15 as having either a cyclin-like or a SAM (Sterile Alpha Motif) domain-like fold.2 Examples include cyclin (1VIN, 1BU2), the TFIIB core domain (1TFB, 1AIS), Recombinase XerD (1A0P) or the RuvA middle domain (1BVS). The structural alignments between NMA1147 and these domains reveal that functional residues in the structural homologues do not align with residues of similar type in NMA1147, nor do they align with the conserved residues in the TPR family [Fig. 1(c)]. Taken together, our analyses thus suggest that the fold of NMA1147 may represent a new SCOP family, or, considering the low sequence identity detected between NMA1147 and its structural homologues, a new SCOP superfamily. Members of this new (super)family may possibly function to mediate protein-protein interactions in a manner that is distinct from that of the classical TPR motif. 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.