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

Abstract

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...

Ab initio determination of the shape of membrane proteins in a nanodisc

“…These complications can be softened by the use of small angle x-ray scattering (SAXS) (Skar-Gislinge et al, 2010;Berthaud et al, 2012;Kynde et al, 2014;Dadinova et al, 2020), which, despite that it only provides a resolution down to about 10 Å, allows one to study complex biological samples directly in solution. Moreover, in recent years, nanodiscs (NDs), i.e.…”

Ab-initio determination of the shape of membrane proteins in a nanodisc

“…These complications can be softened by the use of small angle x-ray scattering (SAXS) (Skar-Gislinge et al, 2010;Berthaud et al, 2012;Kynde et al, 2014;Dadinova et al, 2020), which, despite that it only provides a resolution down to about 10Å, allows one to study complex biological samples directly in solution. Moreover, in recent years, nanodiscs (NDs), i.e.…”

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

“…Wild-type CBS-PPases, including dhPPase, are homotetrameric [19,20]. The deletion variant, which lacked the catalytic DHHA2 domain, retained the tetrameric structure, as demonstrated by two different approaches-size-exclusion chromatography and analytical ultracentrifugation.…”

“…The first proposed model of the entire CBS-PPase molecule based on these structures also assumed that it is homodimeric [18]. However, more recent studies have demonstrated that CBS-PPase is tetrameric [19,20]. We have subsequently proposed two possible structural models of the domain organization in tetramers [19], but neither of them has been verified experimentally.…”