Crystal structure of a hypothetical protein, TTHA0829 from Thermus thermophilus HB8, composed of cystathionine-β-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains

Nakabayashi, Makoto; Shibata, Naoki; Emi, Ishido-Nakai; Kanagawa, Motoi; Iio, Yota; Kakemoto, Hirofumi; Ueda, Y.; Nakagawa, Noriko; Kuramitsu, Seiki; Higuchi, Yoshiki

doi:10.1007/s00792-016-0817-y

Cited by 4 publications

(3 citation statements)

References 58 publications

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“…The three enzymes studied by us were expressed from different bacterial species, but they all form tetramers in solution. The presence of the DRTGG domain in dh-PPase and its absence in el-PPase and eh-PPase suggests that this domain does not participate in the formation of the tetramer, while the regulatory insert promotes oligomerization [13,39].…”

Section: Discussionmentioning

confidence: 99%

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

et al. 2020

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Quaternary structure of CBS-pyrophosphatases (CBS-PPases), which belong to the PPases of family II, plays an important role in their function ensuring cooperative behavior of the enzymes. Despite an intensive research, high resolution structures of the full-length CBS-PPases are not yet available making it difficult to determine the signal transmission path from the regulatory to the active center. In the present work, small-angle X-ray scattering (SAXS) combined with size-exclusion chromatography was applied to determine the solution structures of the full-length wild-type CBS-PPases from three different bacterial species. Previously, in the absence of an experimentally determined full-length CBS-PPase structure, a homodimeric model of the enzyme based on known crystal structures of the CBS domain and family II PPase without this domain has been proposed. Our SAXS analyses demonstrate, for the first time, the existence of stable tetramers in solution for all studied CBS-PPases from different sources. Our findings show that further studies are required to establish the functional properties of these enzymes. This is important not only to enhance our understanding of the relation between CBS-PPases structure and function under normal conditions but also because some human pathogens harbor this class of enzymes.Biomolecules 2020, 10, 564 2 of 12 is located at the N-terminal domain. Importantly, substrate binding to the C-terminal domain in its open conformation causes the domain closure [7].About one-quarter of family II PPase enzymes contains a 250-residue insertion within the N-terminal domain [8]. The insertion consists of the DRTGG domain, named after its conserved Asp-Arg and Thr-Gly-Gly motifs (about 120 amino acids)), and two so-called CBS domains, named after cystathionine-β-synthase, where they were identified for the first time. These PPases are called CBS-PPases to distinguish them from common family II PPases. CBS domains bind adenine nucleotides with various affinities, acting as sensors of the cellular energy status [9].Quaternary structure of CBS-pyrophosphatases plays an important role in their activity and contributes to the thermal stability of these enzymes. It is known that the PPases are more active in the dimeric form with four CBS domains than in the monomeric state, exhibiting positive kinetic cooperativity, which is lost upon CBS domains removal [10,11]. The CBS domains, in turn, contribute to aggregation of the PPases in solution. That is why the crystal structure of human cystathionine-β-synthase was only solved for the protein variant lacking the CBS domains [12]. This protein was found to be dimeric in the crystal and it was speculated that the CBS pair may prompt tetramerization of the full-length protein in solution [13], however, no experimental evidence for the tetramerization has been obtained so far. It is worth emphasizing that, despite fairly intensive structural studies of the family II PPases, only a few high resolution three-dimensional structures of family II PPases are avai...

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Section: Discussionmentioning

confidence: 99%

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

et al. 2020

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“…In homotetrameric putative acetoin dehydrogenase TTHA0829 from Thermus thermophilus, the constituting domains have opposite roles, compared to the CBS-PPase models. Its core domain (aspartate-kinase chorismate-mutase tyrA), together with CBS2 domain, participates only in dimer formation, like the CBS and DRTGG domains of CBS-PPase model, whereas two dimers interact only through CBS1 domains that have -helical and loop extensions [32]. Homooctameric inosine monophosphate dehydrogenase is a dimer of plane tetramers and exists in two forms with different contacts between the tetramers -either predominantly between the catalytic domains or predominantly between the regulatory CBS domains [33].…”

Section: Discussionmentioning

confidence: 99%

The Tetrameric Structure of Nucleotide-regulated Pyrophosphatase and Its Modulation by Deletion Mutagenesis and Ligand Binding

Anashkin

Salminen

Орлов

et al. 2020

Preprint

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A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200-250-kDa homotetramers, which seems unprecedented among the four PPase families. The tetrameric structure is stabilized by Co 2+ , the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co 2+ depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation.

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“…To the best of our knowledge, identical architecture has not been observed in other tetrameric CBS-proteins. An "inverted" type of linear homotetramer was found in Mycobacterium tuberculosis cystathionine β-synthase [28] (7XNZ) and the protein THA0829 from Thermus thermophilus HB8 [29] (5AWE) with four CBS modules in the central part and two peripheral catalytic domain dimers on both sides. The basal form of IMP dehydrogenase is a planar tetramer in which only catalytic domains interact with one another [30] (1ZFJ), but ATP and GTP induce the combination of two planar tetramers into an octamer along the fourfold axis via intertwined interactions of both catalytic and CBS domains [31][32][33] (3DQW, 7OJ1, and 7PJI, respectively).…”

Section: D Structure Of Dhppasementioning

confidence: 99%

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain

Zamakhov,

Anashkin,

Moiseenko

et al. 2023

IJMS

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Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A “linear” 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect.

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Crystal structure of a hypothetical protein, TTHA0829 from Thermus thermophilus HB8, composed of cystathionine-β-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains

Cited by 4 publications

References 58 publications

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

Tetrameric Structures of Inorganic CBS-Pyrophosphatases from Various Bacterial Species Revealed by Small-Angle X-ray Scattering in Solution

The Tetrameric Structure of Nucleotide-regulated Pyrophosphatase and Its Modulation by Deletion Mutagenesis and Ligand Binding

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain

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