NMR structure of the conserved hypothetical protein TM0979 from <i>Thermotoga maritima</i>

Peti, Wolfgang; Herrmann, Torsten; Zagnitko, Olga; Grzechnik, Slawek K.; Wüthrich, Kurt

doi:10.1002/prot.20352

Cited by 5 publications

(7 citation statements)

References 25 publications

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“…All three programs confirm, with high confidence limits, that the dimeric structure is physically reasonable, with interface characteristics that are typical of and consistent with the observed affinity. The monomeric Tm0979 structure was determined using a slightly different construct, in a different buffer, and the secondary structural elements were less well defined than for the dimer structure; no additional biochemical data were reported . Since we also find that Tm0979 can populate a monomer at decreased protein concentration, the difference in the results is not very pronounced and may reasonably arise from the somewhat different experimental conditions.…”

Section: Discussionmentioning

confidence: 90%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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 − .…”

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confidence: 99%

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

et al. 2009

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Although the majority of natural proteins exist as protein-protein complexes, the molecular basis for the formation and regulation of such interactions and the evolution of protein interfaces remain poorly understood. We have investigated these phenomena by characterizing the thermal and chemical denaturation of thermophilic DsrEFH proteins that have a common subunit fold but distinct quaternary structures: homodimeric Tm0979 and homotrimeric Mth1491. Tm0979 forms a moderate affinity dimer, and a monomeric intermediate is readily populated at equilibrium and during folding kinetics. In contrast, the Mth1491 trimer has extremely high stability, so that a monomeric form is not measurably populated at equilibrium, although it may be during folding kinetics. A common mechanism for evolution of quaternary structures may be facile formation of a relatively stable monomeric species, with stabilizing intermolecular interactions centering on alternative environments for a beta-strand at the edge of the monomer, augmented by malleable hydrophobic interactions. The exceptional trimer stability arises from a remarkably slow unfolding rate constant, 6.5 x 10(-13) s(-1), which is a common characteristic of highly stable thermophilic and/or oligomeric proteins. The folding characteristics of Tm0979 and Mth1491 have interesting implications for assembly and regulation of homo- and heterooligomeric proteins in vivo.

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

et al. 2009

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“…Moreover, the strict conservation of proline in position 63 is likely to have a role in properly arranging C62 in the active site of Mtb SufT ( Fig 1 ). The overall arrangement of D, E, T, C, and P residues can therefore create an environment to bind a Fe ion or a Fe-S cluster ( Fig 1 ) [ 31 ], although, a well-recognized Fe-S cluster binding motif could not be identified. The structural feature of Mtb SufT, along with its organization within the suf operon, suggests that SufT might have a role in Fe-S cluster maturation.…”

Section: Resultsmentioning

confidence: 99%

“…thaliana HCF101 binds 4Fe-4S cluster and transfers the cluster to an acceptor apo-protein akin to an Fe-S cluster carrier [ 51 ]. However, Mtb SufT contains only one strictly conserved cysteine, whereas Fe-S carriers generally contain two or more cysteine residues for Fe-S cluster coordination [ 31 ]. Consistent with this, we found that Mtb SufT does not bind Fe or Fe-S cluster, rather the conserved cysteine residue is essential for establishing interaction of SufT with SufS, SufU, Acn, and SufR.…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo

Tripathi¹,

Anand²,

Das³

et al. 2022

PLoS Pathog

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Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.

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Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost

Peti

Page

2007

Protein Expression and Purification

194

135

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NMR structure of the conserved hypothetical protein TM0979 from Thermotoga maritima

Cited by 5 publications

References 25 publications

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

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

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation, metabolism, and survival in vivo

Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost

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