A novel member of the YchN‐like fold: Solution structure of the hypothetical protein Tm0979 from Thermotoga maritima

Abstract

We report herein the NMR structure of Tm0979, a structural proteomics target from Thermotoga maritima. The Tm0979 fold consists of four ␤/␣ units, which form a central parallel ␤-sheet with strand order 1234. The first three helices pack toward one face of the sheet and the fourth helix packs against the other face. The protein forms a dimer by adjacent parallel packing of the fourth helices sandwiched between the two ␤-sheets. This fold is very interesting from several points of view. First, it represents the first structure determination for the DsrH family of conserved hypothetical proteins, which are involved in oxidation of intracellular sulfur but have no defined molecular function. Based on structure and sequence analysis, possible functions are discussed. Second, the fold of Tm0979 most closely resembles YchN-like folds; however the proteins that adopt these folds differ in secondary structural elements and quaternary structure. Comparison of these proteins provides insight into possible mechanisms of evolution of quaternary structure through a simple mechanism of hydrophobicity-changing mutations of one or two residues. Third, the Tm0979 fold is found to be similar to flavodoxin-like folds and ␤/␣ barrel proteins, and may provide a link between these very abundant folds and putative ancestral half-barrel proteins.

Folding and Association of Thermophilic Dimeric and Trimeric DsrEFH Proteins: Tm0979 and Mth1491

“…These proteins have similar subunit folds (Figure 1) but very different sequences (17% identity), quaternary structures (Figure 1), and folding characteristics. Previous NMR and preliminary SEC and light scattering data had indicated that Tm0979 can form a micromolar affinity dimer (23) or a monomer (24) in solution. In order to investigate this further, we analyzed the characteristics of the dimer solution structure by interface analysis programs, DiMoVo (50), PISA (51), and NOXclass (52).…”

“…Protein Expression and Purification. Tm0979 and Mth1491 were prepared using pET15b/BL21(GoldλDE3) expression systems, as described previously (23,28), with the following modifications. For Mth1491, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), and 10% (v/v) glycerol were added after elution of the protein from the Ni affinity column to minimize aggregation.…”

“…Mth1491 contains an additional C-terminal β-strand, located at the edge of the subunit parallel to the first strand and at the center of the trimeric structure; in contrast, the C-terminus of Tm0979 is somewhat shorter and poorly structured on the protein surface. Tm0979 can form a monomeric or a dimeric structure of moderate (micromolar) affinity , ; no other stability or subunit affinity data are available for DsrEFH proteins. Other family members exhibit additional quaternary structure variations: homohexameric or heterohexameric, which are dimers of homo- or heterotrimers with central C-terminal β-strands − .…”

Structural and Molecular Genetic Insight into a Widespread Sulfur Oxidation Pathway

“…However, DsrH also fits into the DsrE/F family. Structural information on representatives of the DsrH family of proteins is available through the work of Shin et al on YchN from E. coli, 15 Gaspar et al on Tm0979 from Thermotoga maritima, 16 Christendat et al on MTH1491 from Methanobacterium thermoautotrophicum, 17 and Numata et al on E. coli TusBCD. 18 In contrast to DsrEFH and TusBCD, all others form homooligomers.…”

A Structural Analysis of the Mode of Action of ORF1 in Pseudaminobacter salicylatoxidans: Prediction of its Role in the Global Sulfur Oxidation Operon (sox)

“…This arrangement produces a three-stranded twisted parallel -sheet flanked by helices on both faces. This type of structural arrangement is known as Rossman fold (Gasper et al, 2005). It is shared between diverse families of proteins including dehydrogenases, amidases, nucleotidyl transferases etc (Gasper et al, 2005).…”