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

## TL;DR

This study identifies specific mutations in a protein domain that change how adenosine phosphates regulate the protein's activity and increase binding efficiency.

## Contribution

The paper discovers key residues in CBS domains that control regulatory effects and binding stoichiometry of adenosine phosphates.

## Key findings

- Lys100 replacement reversed ADP's effect from inhibition to activation.
- Lys95 and Gly118 replacements caused ADP to act as both activator and inhibitor depending on concentration.
- Alanine replacements increased mono-adenosine phosphate binding stoichiometry twofold.

## Abstract

Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the “half-of-the-sites” ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a “two non-interacting pairs of interacting regulatory sites” concept in CBS-PPase regulation.