NMR structure of the conserved hypothetical protein TM0979 from Thermotoga maritima

Introduction. The TM0979 gene of Thermotoga maritima encodes a 87-residue conserved hypothetical protein with a molecular weight of 9876 Da and a calculated isoelectric point of 4.7. There is no functional assignment available for this gene. In this manuscript the NMR structure of TM0979 is described. In the context of the Joint Center of Structural Genomics (JCSG) structural proteomics initiative, TM0979 was identified as a potential structural proteomics target on the basis of a highquality one-dimensional (1D) H NMR spectrum. Single crystals were obtained, but the resolution was too low to use X-ray diffraction for the structure determination. The structure determination of TM0979 was therefore pursued using NMR spectroscopy. Based on the resulting threedimensional (3D) structure and the amino acid sequence, we propose that TM0979 is structurally related to the DsrH protein of Chromatium vinosum, which has been implicated in intracellular sulfur oxidation and/or siroheme assembly. The 3D structure of TM0979 (Fig. 1) was determined using standard NMR methods for the sequence-specific backbone and side chain assignments. NOESY peak picking, NOESY peak assignment and 3D structure calculation were performed automatically, using the ATNOS/ CANDID/DYANA software package. Characteristic parameters for the 3D NMR structure determination of TM0979 are summarized in Table I, and a visual display of the molecular structure is afforded by Figure 1. The TM0979 structure contains in the sequence order 1– 1– 2– 2– 3–(310)– 3– 4 four -helices, a 310-helix and a three-stranded parallel -sheet, which is located in the center of the protein and surrounded by the five helices. Heteronuclear N relaxation data show that TM0979 is a rigid protein with intermolecular fluctuations limited to the sub-ns time scale. Only a surface-exposed loop centered on residue E40 and immediately following helix 2, shows increased mobility. Additionally, the Cterminal pentapeptide segment shows increased mobility, as indicated by reduced N{H}-NOE values (data not shown). On the level of the amino acid sequence, TM0979 is closely related with the DsrH protein of Chromatium vinosum, which is encoded as part of a large operon involved in the sulfur dissimilatory oxidation pathway. Although many genes of this operon have been functionally assigned, the three tandem genes encoding the DsrE, DsrF, and DsrH proteins, which are structurally related to each other, have no attached function as yet. Remarkably, the dsrEFH cluster as well as the gene order are conserved in proteobacteria and in some methanogenic archaea such as E. coli (genes yheNML) and Methanobacterium thermoautotrophicum (MTH1357,1358,1360). According to Pott and Dahl, the tentative assignment of these proteins to the siroheme assembly is compromised by their apparent absence in Archaeoglobus fulgidus, an organism that contains a dissimilatory siroheme sulfite reductase. However, the presence of related genes in E. coli, H. influenzae, and methanogenic archaea, which all are organisms that do not contain dissimilatory sulfite reductases but can synthesize assimilatory siroheme sulfite and nitrite reductases, is consistent with the possible involvement of the dsrEFH-encoded polypeptides in siroheme-dependent pathways. However, there is no indication of either dissimilatory sulfite reductase or a siroheme biosynthesis pathway in the T. maritima genome. Therefore, in general, the functional roles of the DsrEFH proteins, including TM0979 (DsrH), remain to be elucidated. Interestingly, the genomic neighborhood of the yheNML cluster in E.coli and in other proteobacteria includes a peptidyl-prolyl cis–trans isomerase and several other proteins related to the translation machinery. TM0979 is the only close homolog of dsrEFH proteins