Solution structure of the hypothetical protein Mth677 from Methanobacterium thermoautotrophicum: a novel alpha+beta fold

Abstract

The structure of Mth677, a hypothetical protein from Methanobacterium thermoautotrophicum (Mth), has been determined by using heteronuclear nuclear magnetic resonance (NMR) methods on a double-labeled (15)N-(13)C sample. Mth677 adopts a novel alpha+beta fold, consisting of two alpha-helices (one N terminal and one C terminal) packed on the same side of a central beta-hairpin. This structure is likely shared by its three orthologs, detected in three other Archaebacteria. There are no clear features in the sequences of these proteins or in the genome organization of Mth to make a reliable functional assignment to this protein. However, the structural similarity to Escherichia coli MinE, the protein which controls that division occurs at the midcell site, lends support to the proposal that Mth677 might be, in Mth, the counterpart of the topological specificity domain of MinE in E. coli.

Figure 1.

Alignment of Mth677 amino acid sequence with those of the other three known orthologs. Strict conservation, property conservation (hydrophobicity or charge), and conservation in at least three sequences are indicated by black, dark gray, and light gray boxes, respectively. Above the sequence, the numbering and determined secondary structure of Mth677 is indicated: the two helices by black boxes and the β-strands by arrows.

Figure 2.

Summary of observed sequential and short-range NOEs, consensus chemical shift index (Wishart and Sykes 1994), and amide proton exchange data at 298 K and pH 7.0 vs. sequence number. The thickness of the sequential NOEs is a qualitative indication of their intensity. In the exchange plot, an open circle indicates that the amide proton of that residue remains in D₂O solution at pH 7 and T 298 K at least for 45 min, and a filled circle that the corresponding HN exchange took between 45 min and more than 15 h.

Figure 3.

Stereo views of the solution structure of Mth677. (Top) Superposition (N, C^α, C^′) of the 30 nuclear magnetic resonance (NMR) conformers showing the backbone in dark green, the hydrophobic core in pale green, the basic side chains in dark blue, the acidic ones in red, and the rest in cyan. (Bottom) View of Mth677 along its longitudinal axis where the predominantly acidic exterior of the protein can be seen. The figure was prepared with the program MolMol (Koradi et al. 1996).

Figure 4.

Comparison between the structures of bacteriophage λ protein W (top) and Mth677 (bottom). A 180° rotation along the longitudinal axis of the molecule is indicated to show that the arrangement of secondary structure elements in both proteins is different, and that they are mirror images. The figure was prepared with the program MolMol (Koradi et al. 1996).

Figure 5.

Comparison between the structures of Escherichia coli MinE^TSD (top) and Mth Mth677 (bottom). MinE^TSD is a noncovalent dimeric protein, in which the chain termini of each monomer have been labeled with primed or unprimed letters. The secondary structure elements that match the ones in Mth677 (one hairpin of one monomer and the helix of each of the two monomers) are colored as in Mth677, and the rest of the chains are gray.

Figure 6.

Comparison between the electrostatic potential surfaces of MinE^TSD and Mth677. A highly negative electrostatic potential is common to both molecules on the solvent-exposed surface of the hairpin(s), shown at the left. This orientation is the same as in Figure 3 ▶.

Figure 7.

One bond ¹H–¹⁵N single quantum correlation of nuclei ¹H and ¹⁵N in Mt677 with assignments.