@article{MTMT:34232792, title = {Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein.}, url = {https://m2.mtmt.hu/api/publication/34232792}, author = {Simon, Márton and Iordanov, Iordan and Szöllősi, András and Csanády, László}, doi = {10.7554/eLife.90736}, journal-iso = {ELIFE}, journal = {ELIFE}, volume = {12}, unique-id = {34232792}, issn = {2050-084X}, abstract = {CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide-binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of ~200-fold. Here, we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed ~15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated ~15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.}, keywords = {ZEBRAFISH; Xenopus; molecular biophysics; Structural biology; D-loop; composite ATP-binding site; flickery closure; mutant cycle}, year = {2023}, eissn = {2050-084X}, orcid-numbers = {Iordanov, Iordan/0000-0001-8251-5857; Szöllősi, András/0000-0002-5570-4609; Csanády, László/0000-0002-6547-5889} } @article{MTMT:34064647, title = {Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer}, url = {https://m2.mtmt.hu/api/publication/34064647}, author = {Szöllősi, András and Almássy, János}, doi = {10.1038/s41598-023-38640-6}, journal-iso = {SCI REP}, journal = {SCIENTIFIC REPORTS}, volume = {13}, unique-id = {34064647}, issn = {2045-2322}, abstract = {Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca 2+ , ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca 2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (P o ) shows little voltage dependence and is stable over several minutes. P o is high without addition of exogenous PIP 2 , but is largely blunted by treatment with poly- l -Lysine, a polycation that masks PIP 2 headgroups. These results indicate that PIP 2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP 2 binding affinity of nvTRPM2 under physiological conditions.}, year = {2023}, eissn = {2045-2322}, orcid-numbers = {Szöllősi, András/0000-0002-5570-4609} } @article{MTMT:32040119, title = {Two decades of evolution of our understanding of the transient receptor potential melastatin 2 (Trpm2) cation channel}, url = {https://m2.mtmt.hu/api/publication/32040119}, author = {Szöllősi, András}, doi = {10.3390/life11050397}, journal-iso = {LIFE-BASEL}, journal = {LIFE-BASEL}, volume = {11}, unique-id = {32040119}, abstract = {The transient receptor potential melastatin (TRPM) family belongs to the superfamily of TRP ion channels. It consists of eight family members that are involved in a plethora of cellular functions. TRPM2 is a homotetrameric Ca2+-permeable cation channel activated upon oxidative stress and is important, among others, for body heat control, immune cell activation and insulin secretion. Invertebrate TRPM2 proteins are channel enzymes; they hydrolyze the activating ligand, ADP-ribose, which is likely important for functional regulation. Since its cloning in 1998, the understanding of the biophysical properties of the channel has greatly advanced due to a vast number of structure– function studies. The physiological regulators of the channel have been identified and characterized in cell-free systems. In the wake of the recent structural biochemistry revolution, several TRPM2 cryo-EM structures have been published. These structures have helped to understand the general features of the channel, but at the same time have revealed unexplained mechanistic differences among channel orthologues. The present review aims at depicting the major research lines in TRPM2 structure-function. It discusses biophysical properties of the pore and the mode of action of direct channel effectors, and interprets these functional properties on the basis of recent three-dimensional structural models. © 2021 by the author. Licensee MDPI, Basel, Switzerland.}, keywords = {ION CHANNELS; ADP-ribose; Single particle cryo-EM; TRPM2; Nudix hydrolase}, year = {2021}, eissn = {2075-1729}, orcid-numbers = {Szöllősi, András/0000-0002-5570-4609} } @article{MTMT:30637149, title = {Enzyme activity and selectivity filter stability of ancient TRPM2 channels were simultaneously lost in early vertebrates}, url = {https://m2.mtmt.hu/api/publication/30637149}, author = {Iordanov, Iordan and Tóth, Balázs and Szöllősi, András and Csanády, László}, doi = {10.7554/eLife.44556}, journal-iso = {ELIFE}, journal = {ELIFE}, volume = {8}, unique-id = {30637149}, issn = {2050-084X}, abstract = {Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel important for the immune response, insulin secretion, and body temperature regulation. It is activated by cytosolic ADP ribose (ADPR) and contains a nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain homologous to ADPR phosphohydrolases (ADPRases). Human TRPM2 (hsTRPM2) is catalytically inactive due to mutations in the conserved Nudix box sequence. Here, we show that TRPM2 Nudix motifs are canonical in all invertebrates but vestigial in vertebrates. Correspondingly, TRPM2 of the cnidarian Nematostella vectensis (nvTRPM2) and the choanoflagellate Salpingoeca rosetta (srTRPM2) are active ADPRases. Disruption of ADPRase activity fails to affect nvTRPM2 channel currents, reporting a catalytic cycle uncoupled from gating. Furthermore, pore sequence substitutions responsible for inactivation of hsTRPM2 also appeared in vertebrates. Correspondingly, zebrafish (Danio rerio) TRPM2 (drTRPM2) and hsTRPM2 channels inactivate, but srTRPM2 and nvTRPM2 currents are stable. Thus, catalysis and pore stability were lost simultaneously in vertebrate TRPM2 channels.}, keywords = {Xenopus; E. coli; molecular biophysics; Structural biology; Selectivity filter; ADP ribose; Nudix hydrolase; channel enzyme; rundown}, year = {2019}, eissn = {2050-084X}, orcid-numbers = {Iordanov, Iordan/0000-0001-8251-5857; Tóth, Balázs/0000-0002-1257-2597; Szöllősi, András/0000-0002-5570-4609; Csanády, László/0000-0002-6547-5889} } @article{MTMT:30465914, title = {Structure of a TRPM2 channel in complex with Ca2+ explains unique gating regulation}, url = {https://m2.mtmt.hu/api/publication/30465914}, author = {Zhang, Zhe and Tóth, Balázs and Szöllősi, András and Chen, Jue and Csanády, László}, doi = {10.7554/eLife.36409}, journal-iso = {ELIFE}, journal = {ELIFE}, volume = {7}, unique-id = {30465914}, issn = {2050-084X}, abstract = {Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel required for immune cell activation, insulin secretion, and body heat control. TRPM2 is activated by cytosolic Ca2+, phosphatidyl-inositol-4,5-bisphosphate and ADP ribose. Here, we present the 3 A resolution electron cryo-microscopic structure of TRPM2 from Nematostella vectensis, 63% similar in sequence to human TRPM2, in the Ca2+-bound closed state. Compared to other TRPM channels, TRPM2 exhibits unique structural features that correlate with its function. The pore is larger and more negatively charged, consistent with its high Ca2+ selectivity and larger conductance. The intracellular Ca2+ binding sites are connected to the pore and cytosol, explaining the unusual dependence of TRPM2 activity on intra- and extracellular Ca2+. In addition, the absence of a post filter motif is likely the cause of the rapid inactivation of human TRPM2. Together, our cryo-EM and electrophysiology studies provide a molecular understanding of the unique gating mechanism of TRPM2.}, year = {2018}, eissn = {2050-084X}, orcid-numbers = {Tóth, Balázs/0000-0002-1257-2597; Szöllősi, András/0000-0002-5570-4609; Csanády, László/0000-0002-6547-5889} } @article{MTMT:3110723, title = {Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter}, url = {https://m2.mtmt.hu/api/publication/3110723}, author = {Szöllősi, András and Vieira-Pires, Ricardo S and Teixeira-Duarte, Celso M and Rocha, R and Morais-Cabral, Joao H}, doi = {10.1371/journal.pbio.1002356}, journal-iso = {PLOS BIOL}, journal = {PLOS BIOLOGY}, volume = {14}, unique-id = {3110723}, issn = {1544-9173}, abstract = {KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K+ and Na+) transport proteins that closely resemble K+ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K+ channels. © 2016 Szollosi et al.}, keywords = {ARTICLE; membrane protein; controlled study; nonhuman; Protein Conformation; unclassified drug; protein expression; ion transport; Adenosine Triphosphate; protein purification; protein protein interaction; molecular dynamics; structure activity relation; potassium channel; structure analysis; protein structure; protein tyrosine kinase; cation transport protein; proteoliposome; KtrB potassium ion transporter; KtrA potassium ion transporter}, year = {2016}, eissn = {1545-7885}, orcid-numbers = {Szöllősi, András/0000-0002-5570-4609} } @article{MTMT:2333381, title = {Conformational changes in the catalytically inactive nucleotide-binding site of CFTR}, url = {https://m2.mtmt.hu/api/publication/2333381}, author = {Csanády, László and Mihályi, Csaba and Szöllősi, András and Törőcsik, Beáta and Vergani, P}, doi = {10.1085/jgp.201210954}, journal-iso = {J GEN PHYSIOL}, journal = {JOURNAL OF GENERAL PHYSIOLOGY}, volume = {142}, unique-id = {2333381}, issn = {0022-1295}, abstract = {A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C-->O1-->O2-->C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2x) transition O1-->O2 and decreases the nonhydrolytic closing rate (transition O1-->C) of CFTR mutants K1250A ( approximately 4x) and E1371S ( approximately 3x). Mutation H1348A also slowed ( approximately 3x) the O1-->O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A ( approximately 3x) and E1371S ( approximately 3x) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1-->C and O1-->O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.}, year = {2013}, eissn = {1540-7748}, pages = {61-73}, orcid-numbers = {Csanády, László/0000-0002-6547-5889; Mihályi, Csaba/0000-0001-7536-3066; Szöllősi, András/0000-0002-5570-4609; Törőcsik, Beáta/0000-0002-9838-3710} } @article{MTMT:2329285, title = {The structure of the KtrAB potassium transporter}, url = {https://m2.mtmt.hu/api/publication/2329285}, author = {Vieira-Pires, RS and Szöllősi, András and Morais-Cabral, JH}, doi = {10.1038/nature12055}, journal-iso = {NATURE}, journal = {NATURE}, volume = {496}, unique-id = {2329285}, issn = {0028-0836}, abstract = {In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na + or K + channels and possibly as cation/K + symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K + transporter, the KtrAB complex from Bacillus subtilis. The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB-ATP and the structures of the isolated full-length KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters. © 2013 Macmillan Publishers Limited. All rights reserved.}, keywords = {PH; PROTEIN; LIGAND; ARTICLE; POTASSIUM; Models, Biological; Protein Subunits; amino acid sequence; membrane protein; Escherichia coli; priority journal; Osmosis; Cytoplasm; nonhuman; structure-activity relationship; Models, Molecular; Protein Conformation; Homeostasis; ION; Cytosol; unclassified drug; ion transport; Adenosine Triphosphate; crystal structure; bacterium; protein protein interaction; Crystallography, X-Ray; complex formation; BIOCHEMISTRY; ligand binding; Bacillus subtilis; protein structure; conformational transition; Bacterial Proteins; potassium transport; Cation Transport Proteins; adenosine diphosphate; Functional role; protein KtrB; protein KtrAB; protein KtrA; carrier proteins and binding proteins}, year = {2013}, eissn = {1476-4687}, pages = {323-328}, orcid-numbers = {Szöllősi, András/0000-0002-5570-4609} } @article{MTMT:1690126, title = {Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating}, url = {https://m2.mtmt.hu/api/publication/1690126}, author = {Szöllősi, András and Muallem, DR and Csanády, László and Vergani, P}, doi = {10.1085/jgp.201110608}, journal-iso = {J GEN PHYSIOL}, journal = {JOURNAL OF GENERAL PHYSIOLOGY}, volume = {137}, unique-id = {1690126}, issn = {0022-1295}, year = {2011}, eissn = {1540-7748}, pages = {549-562}, orcid-numbers = {Szöllősi, András/0000-0002-5570-4609; Csanády, László/0000-0002-6547-5889} } @article{MTMT:1504152, title = {Pharmacological stimulation and inhibition of insulin secretion in mouse islets lacking ATP-sensitive K plus channels}, url = {https://m2.mtmt.hu/api/publication/1504152}, author = {Szöllősi, András and Nenquin, M and Henquin, JC}, doi = {10.1111/j.1476-5381.2009.00588.x}, journal-iso = {BR J PHARMACOL}, journal = {BRITISH JOURNAL OF PHARMACOLOGY}, volume = {159}, unique-id = {1504152}, issn = {0007-1188}, year = {2010}, eissn = {1476-5381}, pages = {669-677}, orcid-numbers = {Szöllősi, András/0000-0002-5570-4609} }