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

Abstract

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.

The inorganic pyrophosphatases of microorganisms: a structural and functional review

“…Additionally, Zamakhov et al (2023) confirmed the tetrameric nature of these enzymes by purifying and characterizing the enzyme of Desulfobacterium hafniense by transmission electron microscopy and biochemical approaches. They found that a highly flexible region is responsible for limiting the crystallization of these enzymes, which is consistent with the findings of G. intestinalis .…”

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase

“…Unlike the canonical Family II PPases, CBS-PPases form tetramers [ 11 ], which are stabilized by separate crosswise interactions of the catalytic and regulatory parts [ 12 ] ( Figure 1 B). The structure of tetrameric CBS-PPase from Desulfitobacterium hafniense ( dh PPase) without the DHHA2 domain was obtained at 16 Å resolution by single-particle electron microscopy [ 12 ], whereas the structures of the dimeric regulatory part of homologous Clostridium perfringens CBS-PPase ( cp PPase) in complexes with AMP (inhibitor) and Ap 4 A (activator) were determined at 2.3 Å by X-ray crystallography [ 13 ]. Although each CBS domain can bind a mono-adenosine phosphate and there are two CBS domains per subunit, the structures indicated the binding stoichiometry of one molecule of AMP per subunit or one molecule of Ap 4 A per dimer [ 10 , 13 ].…”

“… ( A ) Domain composition of canonical PPase and CBS-PPase of Family II. ( B ) 3D structure of tetrameric dh PPase without the DHHA2 domain suggested by low-resolution cryo-EM and molecular modeling [ 12 ]. The four subunits, a1–a4, are shown in different colors.…”

“…The region of the active site retained in the truncated subunit is indicated by a red circle and is the location where the missing DHHA2 domain is attached. Panel B was reproduced from Zamakhov et al [ 12 ] under the Creative Commons CC BY license.…”