title: The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.
abstract: Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies.
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.|MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary.
authors.forename: Iordan
authors.identifier: 0000-0001-8251-5857
authors.initials: I
authors.lastname: Iordanov
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.|MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary.
authors.forename: Csaba
authors.identifier: 0000-0001-7536-3066
authors.initials: C
authors.lastname: Mihályi
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.|MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Tóth
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary.|MTA-SE Ion Channel Research Group, Semmelweis University, Budapest, Hungary.
authors.forename: László
authors.identifier: 0000-0002-6547-5889
authors.initials: L
authors.lastname: Csanády
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D000246:Adenosine Diphosphate Ribose; D006867:Hydrolases
keywords: ADPR; E. coli; NUDT9H domain; TRPM2; biochemistry; biophysics; chanzyme; chimera; structural biology; xenopus