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

Author:

Zamakhov Ilya M.12,Anashkin Viktor A.1ORCID,Moiseenko Andrey V.3,Orlov Victor N.1ORCID,Vorobyeva Natalia N.1,Sokolova Olga S.34ORCID,Baykov Alexander A.1ORCID

Affiliation:

1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia

2. Department of Chemistry, Lomonosov Moscow State University, 119899 Moscow, Russia

3. Department of Biology, Lomonosov Moscow State University, 119899 Moscow, Russia

4. Department of Biology, Shenzhen MSU-BIT University, Shenzhen 518172, China

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.

Funder

Russian Science Foundation

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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