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bioRxiv · 10.1101/2020.04.07.028951

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

Anashkin, V. A.·Salminen, A.·Orlov, V. N.·Lahti, R.·Baykov, A. A.
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Abstract

A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine {beta}-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 Co2+, 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. Co2+ 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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Anashkin, V. A., Salminen, A., Orlov, V. N., Lahti, R., Baykov, A. A.. 2020-04-07. The Tetrameric Structure of Nucleotide-regulated Pyrophosphatase and Its Modulation by Deletion Mutagenesis and Ligand Binding. https://doi.org/10.1101/2020.04.07.028951

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