An arginine residue involved in allosteric regulation of cystathionine β-synthase (CBS) domain-containing pyrophosphatase

Anashkin, Viktor A.; Орлов, В. Н.; Lahti, Reijo; Baykov, Alexander A.

doi:10.1016/j.abb.2018.11.024

Cited by 6 publications

(19 citation statements)

References 24 publications

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“…indicates lack of information. Columns marked as N, M, and D refer to the effects measured without any nucleotide, in the presence of the mononucleotides, and in the presence of the dinucleotides, respectively.bFrom ref (18). cFrom ref (17).…”

Section: Discussionmentioning

confidence: 99%

“…Wild-type dh PPase (UniProtKB: B8FP42) and its variants containing a His 6 tag at their N-termini were isolated from E. coli BL21 cells transformed with the pET-28b vector (Novagen) carrying the corresponding gene, as described previously. 18 Site-directed mutagenesis was performed using overlap extension PCR with Phusion DNA polymerase. The purity of the final products was estimated by SDS-PAGE 32 with Coomassie staining.…”

Section: Methodsmentioning

confidence: 99%

“…Nucleotides effects on activity at a fixed substrate concentration were analyzed in terms of Scheme 1, written in terms of microscopic binding constants, like in our latest publication. 18 Use of microscopic constants simplified analysis in comparison to the use of macroscopic constants in previous analyses 14,15,17 without effect on its results. The microscopic and macroscopic binding (and Michaelis) constants are linked by the following relationships: K N1(micro) = 2 K N1(macro) , K N2(micro) = 0.5 K N2(macro) .…”

Section: Methodsmentioning

confidence: 99%

“…38 The dependences of the hydrolysis rate ( v N ) on nucleotide concentration ([N]) were fit to eq 1. 18 In the framework of Scheme 1, binding cooperativity can be diagnosed by comparing the K N constants: K N1 > K N2 indicates positive cooperativity, K N1 < K N2 indicates negative cooperativity, and K N1 = K N2 is indicative of noncooperative behavior. Decreases in the free nucleotide concentration because of binding to the enzyme could be neglected in all cases, except for the pair R295A variant/Ap 4 A.…”

Section: Methodsmentioning

confidence: 99%

“…17 The arginine located in the CBS2 domain has been found to control kinetic cooperativity, nucleotide-binding affinity, and the size of the regulatory signal exerted by both mono- and dinucleotides. 18 However, transmission of the regulatory signal from the CBS domains to a distantly located catalytic site and between subunits in CBS-PPase should involve an extended residue network that links all catalytic and regulatory sites in CBS-PPase. To identify other members of the network, we performed an extensive mutagenesis study of the subunit interface-forming CBS and DHH domains.…”

Section: Introductionmentioning

confidence: 99%

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Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

et al. 2019

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Inorganic pyrophosphatase containing regulatory cystathionine β-synthase (CBS) domains (CBS-PPase) is inhibited by adenosine monophosphate (AMP) and adenosine diphosphate and activated by adenosine triphosphate (ATP) and diadenosine polyphosphates; mononucleotide binding to CBS domains and substrate binding to catalytic domains are characterized by positive cooperativity. This behavior implies three pathways for regulatory signal transduction — between regulatory and active sites, between two active sites, and between two regulatory sites. Bioinformatics analysis pinpointed six charged or polar amino acid residues of Desulfitobacterium hafniense CBS-PPase as potentially important for enzyme regulation. Twelve mutant enzyme forms were produced, and their kinetics of pyrophosphate hydrolysis was measured in wide concentration ranges of the substrate and various adenine nucleotides. The parameters derived from this analysis included catalytic activity, Michaelis constants for two active sites, AMP-, ATP-, and diadenosine tetraphosphate-binding constants for two regulatory sites, and the degree of activation/inhibition for each nucleotide. Replacements of arginine 295 and asparagine 312 by alanine converted ATP from an activator to an inhibitor and markedly affected practically all the above parameters, indicating involvement of these residues in all the three regulatory signaling pathways. Replacements of asparagine 312 and arginine 334 abolished or reversed kinetic cooperativity in the absence of nucleotides but conferred it in the presence of diadenosine tetraphosphate, without effects on nucleotide-binding parameters. Modeling and molecular dynamics simulations revealed destabilization of the subunit interface as a result of asparagine 312 and arginine 334 replacements by alanine, explaining abolishment of kinetic cooperativity. These findings identify residues 295, 312, and 334 as crucial for CBS-PPase regulation via CBS domains.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

et al. 2019

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Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase

Anashkin

Aksenova

Vorobyeva

et al. 2019

Biochimica et Biophysica Acta (BBA) - General Subjects

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

Anashkin,

Kirillova,

Orlov

et al. 2024

Preprint

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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 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 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 activator at low concentrations but inhibitor at high concentrations. Replacements of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding twofold. These findings identify several key protein residues and suggest “two non-interacting pairs of interacting regulatory sites” concept in CBS-PPase regulation.

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An arginine residue involved in allosteric regulation of cystathionine β-synthase (CBS) domain-containing pyrophosphatase

Cited by 6 publications

References 24 publications

Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides

Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase

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

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