PMC 20140719 pmc.key 4852598 CC BY no 0 0 10.1038/ncomms11337 ncomms11337 4852598 27088325 11337 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ surname:van den Berg;given-names:Bert surname:Chembath;given-names:Anupama surname:Jefferies;given-names:Damien surname:Basle;given-names:Arnaud surname:Khalid;given-names:Syma surname:Rutherford;given-names:Julian C. TITLE front 7 2016 0 Structural basis for Mep2 ammonium transceptor activation by phosphorylation protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:34:03Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:33:50Z ammonium transceptor ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:13:59Z phosphorylation ABSTRACT abstract 77 Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation. protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z transceptors chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:36:41Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:34:16Z Mep2 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:13:59Z phosphorylation evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:21Z X-ray crystal structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:28:54Z Mep2 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:36:35Z Saccharomyces cerevisiae species MESH: melaniev@ebi.ac.uk 2023-03-15T16:36:48Z Candida albicans protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:57Z transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:34Z not phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:11Z inactive protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:28:58Z Mep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:18:42Z cytoplasmic loops structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:56Z C-terminal region structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR site SO: melaniev@ebi.ac.uk 2023-03-16T11:36:27Z exit site SO: melaniev@ebi.ac.uk 2023-03-16T11:36:32Z channel residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:47Z His2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:32:08Z solvent accessible site SO: melaniev@ebi.ac.uk 2023-03-16T11:36:37Z negatively charged pocket site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:44Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:38:37Z phosphorylation-mimicking mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:40:36Z Mep2 variants species MESH: melaniev@ebi.ac.uk 2023-03-15T16:40:10Z C. albicans protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:32:12Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:05Z eukaryotic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:36:54Z ammonium ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:13:59Z phosphorylation ABSTRACT abstract 1224 Mep2 proteins are tightly regulated fungal ammonium transporters. Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria. protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:57:59Z crystal structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T16:46:37Z by comparing them with protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:38Z bacteria INTRO paragraph 1481 Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. A common feature of transceptors is that they are induced when cells are starved for their substrate. While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways. One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter. protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z Transceptors protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:46:25Z membrane proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z transceptors species MESH: melaniev@ebi.ac.uk 2023-03-15T16:36:35Z Saccharomyces cerevisiae protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z transceptors chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:45:39Z phosphate protein PR: melaniev@ebi.ac.uk 2023-03-15T16:45:43Z Pho84 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:45:47Z amino acids protein PR: melaniev@ebi.ac.uk 2023-03-15T16:45:50Z Gap1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:45:56Z ammonium protein PR: melaniev@ebi.ac.uk 2023-03-15T16:45:59Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z transceptors taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:45:06Z higher eukaryotes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:44:57Z mammalian protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:27Z SNAT2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:46:20Z amino-acid transporter protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:30Z GLUT2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:46:16Z glucose transporter protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z transceptors protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:46:12Z transporter INTRO paragraph 2271 Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources. protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:04Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:52:34Z (methylammonium (MA) permease) proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:33Z ammonium transceptors taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:50:14Z fungi protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:52:31Z Amt/Mep/Rh family of transporters taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:52:27Z all kingdoms of life chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:50:22Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:50:14Z Fungi protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:08Z Mep protein PR: melaniev@ebi.ac.uk 2023-03-15T16:52:17Z Mep1-3 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:36:23Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:34Z Mep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:35Z transceptors protein PR: melaniev@ebi.ac.uk 2023-03-15T16:51:43Z Mep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T16:51:49Z Mep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T16:52:13Z Mep1 protein PR: melaniev@ebi.ac.uk 2023-03-15T16:52:05Z Mep3 protein PR: melaniev@ebi.ac.uk 2023-03-15T16:51:56Z Mep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:51:52Z highly expressed chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:51:28Z MA protein PR: melaniev@ebi.ac.uk 2023-03-15T16:51:40Z Mep2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:37:37Z ammonium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:51:17Z nitrogen INTRO paragraph 3393 With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. All structures show the transporters in open conformations. Intriguingly, fundamental questions such as the nature of the transported substrate and the transport mechanism are still controversial. Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport. species MESH: melaniev@ebi.ac.uk 2023-03-15T16:58:24Z human protein PR: melaniev@ebi.ac.uk 2023-03-15T16:58:35Z RhCG evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:00:00Z structure taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:05Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:59:54Z Amt evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:57:59Z crystal structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T16:58:04Z molecular dynamics experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T16:58:11Z MD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:00:06Z structures protein PR: melaniev@ebi.ac.uk 2023-03-15T16:58:35Z RhCG protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:28Z stable oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:57:46Z trimers oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:27Z monomer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:57:08Z transmembrane structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:57:20Z TM structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:57:35Z helices site SO: melaniev@ebi.ac.uk 2023-03-15T17:00:25Z central channel chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T10:48:09Z ammonium evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:57:52Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:12Z transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T11:41:53Z ammonia protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:14Z Amt/Mep proteins protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:44Z active protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T17:00:14Z electrogenic transporters chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:43Z NH4+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:55Z NH3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:59:01Z H+ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:32:17Z highly conserved site SO: melaniev@ebi.ac.uk 2023-03-16T11:32:52Z channel residue_name SO: melaniev@ebi.ac.uk 2023-03-15T17:08:15Z histidine structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:55Z NH3 site SO: melaniev@ebi.ac.uk 2023-03-16T11:36:48Z channel chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:55Z NH3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:59:10Z H+ INTRO paragraph 4562 Ammonium transport is tightly regulated. In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:35:58Z Ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:16Z animals chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:35:30Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:08:43Z microorganisms chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T11:41:58Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:38Z bacteria gene GENE: melaniev@ebi.ac.uk 2023-03-16T11:44:27Z amt gene GENE: melaniev@ebi.ac.uk 2023-03-16T11:44:30Z glnK protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T17:08:38Z PII-like signal transduction class protein protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:21Z Amt proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:25Z transporter protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:31Z GlnK chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:35:42Z ammonium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:38:07Z nitrogen protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:34Z GlnK protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:13:36Z uridylated protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:37Z Amt proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:24Z eukaryotes protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:41Z GlnK chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:38:17Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:27Z plants protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:44Z transporter ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:13:59Z phosphorylation ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:11Z dephosphorylation species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:13:59Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:56Z C-terminal region structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:35:14Z Mep2 INTRO paragraph 5574 To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. The structures are similar to each other but show considerable differences to all other ammonium transporter structures. The most striking difference is the fact that the Mep2 proteins have closed conformations. The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport. protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:38:32Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:21Z X-ray crystal structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T18:40:14Z Mep2 transceptors species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae species MESH: melaniev@ebi.ac.uk 2023-03-15T16:40:10Z C. albicans evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:40:28Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T18:40:11Z ammonium transporter evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:40:31Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:32:59Z solvent accessible site SO: melaniev@ebi.ac.uk 2023-03-15T18:40:07Z negatively charged pocket site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit site SO: melaniev@ebi.ac.uk 2023-03-15T18:41:07Z channels protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:40:53Z phosphorylation-mimicking mutants species MESH: melaniev@ebi.ac.uk 2023-03-15T16:40:10Z C. albicans protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:39Z Mep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:40:43Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:50Z structure mutant MESH: melaniev@ebi.ac.uk 2023-03-15T18:39:45Z Mep2 variant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:38:55Z lacking structure_element SO: melaniev@ebi.ac.uk 2023-03-15T18:38:29Z segment site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:05Z eukaryotic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:38:50Z ammonium RESULTS title_1 6478 Results RESULTS title_2 6486 General architecture of Mep2 ammonium transceptors protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T18:41:31Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:33Z ammonium transceptors RESULTS paragraph 6537 The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. Unless specifically stated, the drawn conclusions also apply to ScMep2. protein PR: melaniev@ebi.ac.uk 2023-03-15T18:53:13Z Mep2 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:51:54Z overexpressed species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:51:57Z structure determination experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:52:00Z X-ray crystallography experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:52:03Z molecular replacement experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:52:07Z MR taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:52:14Z archaebacterial protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:52:30Z structure evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:52:35Z structure protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:52:39Z structure–function studies taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T18:53:21Z Mep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:53:53Z overexpressed and screened for evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:53:57Z crystals protein PR: melaniev@ebi.ac.uk 2023-03-15T18:53:44Z Mep2 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:40:10Z C. albicans protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T18:54:00Z structure determination evidence DUMMY: melaniev@ebi.ac.uk 2023-06-14T09:45:50Z crystal forms protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:54:07Z structures protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:48:17Z r.m.s.d. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:48:45Z root mean square deviation structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:26:42Z Electron density residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:44:36Z 43 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:44:39Z 25 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:39Z poorly conserved protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:51:49Z disordered protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:12Z trimeric oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:27Z monomer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T18:49:54Z TM helices structure_element SO: melaniev@ebi.ac.uk 2023-03-15T18:55:30Z central pore site SO: melaniev@ebi.ac.uk 2023-03-15T18:55:52Z ammonium binding site site SO: melaniev@ebi.ac.uk 2023-03-15T18:56:01Z Phe gate structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif site SO: melaniev@ebi.ac.uk 2023-03-15T18:51:24Z hydrophobic channel taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T18:56:19Z transporters protein PR: melaniev@ebi.ac.uk 2023-03-15T16:58:35Z RhCG protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:55Z structure protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 RESULTS paragraph 8338 While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear. protein PR: melaniev@ebi.ac.uk 2023-03-15T19:01:22Z Mep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:00:53Z prokaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:29:54Z transporters evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:59Z r.m.s.d. protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:37Z intracellular loops structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:44Z ICLs structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:13Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:01:29Z 20–25 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:04:29Z extracellular domain oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:27Z monomer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:55Z extracellular loop structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:02:03Z ECL5 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:27Z monomer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:04:38Z extracellular loops structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:03:25Z vestibule site SO: melaniev@ebi.ac.uk 2023-03-15T18:55:52Z ammonium binding site taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:03:25Z vestibule oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-07-21T08:26:16Z monomer protein PR: melaniev@ebi.ac.uk 2023-03-15T19:02:59Z Mep2 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T19:03:08Z AMT proteins protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:42:36Z deletion mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-15T19:04:22Z 2-27Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:43Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T19:03:02Z Mep2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:39:36Z ammonium protein PR: melaniev@ebi.ac.uk 2023-03-22T09:39:51Z Mep2 RESULTS title_2 9498 Mep2 channels are closed by a two-tier channel block protein PR: melaniev@ebi.ac.uk 2023-03-15T19:05:28Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-15T19:05:39Z channels protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:06Z channel block RESULTS paragraph 9551 The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). ICL1 has also moved inwards relative to its position in the bacterial Amts. The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2). protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:54Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:32:03Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:30:57Z ammonium transporter evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:05:14Z structures protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:45Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:31Z TM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z Amts structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:06:39Z conserved protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:06:42Z basic structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:06:50Z RxK motif residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:03:56Z Arg54 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:03:47Z Lys55 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:04Z Lys56 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:07:07Z basic structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:07:10Z motif protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:49Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:07:01Z structures protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:06:51Z RxK motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:34:46Z TM1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:07:38Z relatively conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:15Z Tyr49 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:24Z Tyr53 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bond protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:53Z absolutely conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:42Z His342 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:33Z His348 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 site SO: melaniev@ebi.ac.uk 2023-03-15T21:07:58Z channel taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:08:23Z Amt proteins residue_name SO: melaniev@ebi.ac.uk 2023-03-15T21:08:06Z Tyr structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:34:46Z TM1 site SO: melaniev@ebi.ac.uk 2023-03-15T21:14:12Z channel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open residue_name SO: melaniev@ebi.ac.uk 2023-03-15T17:08:15Z histidine RESULTS paragraph 11074 Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section). structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:13:11Z ICL2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:13:23Z conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:13:31Z Glu140 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:13:39Z Arg141 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:13Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:13:45Z pseudo-symmetrical halves structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:13:48Z TM1-5 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:13:52Z TM6-10 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:13:57Z transporter site SO: melaniev@ebi.ac.uk 2023-03-15T21:14:05Z channel structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:06Z channel block chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:40:28Z ammonium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:40:40Z nitrogen protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed site SO: melaniev@ebi.ac.uk 2023-03-15T21:14:16Z channel evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:14:52Z no density chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T21:14:30Z ammonium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T21:14:39Z water protein PR: melaniev@ebi.ac.uk 2023-03-15T21:14:25Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-15T21:14:21Z channel structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T21:14:44Z crystallized chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T21:12:00Z ammonium RESULTS paragraph 11967 The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. In the Mep2 structures, none of the interactions mentioned above are present. protein PR: melaniev@ebi.ac.uk 2023-03-15T21:22:19Z Mep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:22:16Z transporters structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR protein PR: melaniev@ebi.ac.uk 2023-03-15T21:22:23Z Mep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:22:36Z main body protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:22:40Z transporter protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:22:44Z elongated evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:22:51Z structures taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:25:08Z N-terminal half protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:23:02Z transporters structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:23:06Z TM1-5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:10Z compact evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:13Z structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:17Z universally conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:25Z Arg415 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:29Z 370 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:37Z Glu421 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:41Z 376 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:47Z Gly424 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:51Z 379 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:59Z Asp426 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:24:04Z 381 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:24:11Z Tyr 435 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:24:16Z 390 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:24:27Z ‘ExxGxD' motif experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T21:24:43Z mutated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:11Z inactive protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:24:51Z transporters protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:05Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:25:08Z N-terminal half residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:25:33Z Tyr390 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:23Z Amt-1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bonded protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:25:41Z conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:25:49Z His185 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:26:04Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:26:18Z Gly172 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:26:26Z Lys173 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bonded residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:26:44Z Arg370 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:41:16Z modelled protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:44Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein PR: melaniev@ebi.ac.uk 2023-03-15T21:27:13Z AtAmt-1;1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:17Z structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:26Z Y467 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:35Z H239 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:42Z D458 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:50Z K71 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:25:08Z N-terminal half protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:27:00Z transporters residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:28:10Z Asp381 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:31Z TM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters protein PR: melaniev@ebi.ac.uk 2023-03-15T21:29:23Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:29:26Z structures RESULTS title_2 13717 Phosphorylation target site is at the periphery of Mep2 site SO: melaniev@ebi.ac.uk 2023-03-15T21:31:00Z Phosphorylation target site protein PR: melaniev@ebi.ac.uk 2023-03-15T21:31:10Z Mep2 RESULTS paragraph 13773 Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3). residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:48Z phosphorylated protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:35:57Z TORC1 effector kinase protein PR: melaniev@ebi.ac.uk 2023-03-15T21:36:03Z Npr1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:41:18Z nitrogen protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:36:25Z absence of protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:54Z Npr1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T21:36:33Z plasmid-encoded protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T21:36:40Z Mep2 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:36:44Z mep1-3Δ mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:26Z triple mepΔ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T21:36:52Z ammonium protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:36:57Z transporter protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:11Z inactive protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:38:37Z phosphorylation-mimicking mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:14Z S457D protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:44Z active mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:26Z triple mepΔ mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:41Z triple mepΔ npr1Δ experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:47Z Mutation site SO: melaniev@ebi.ac.uk 2023-03-15T21:38:02Z phosphorylation sites structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:38:12Z npr1Δ ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 protein PR: melaniev@ebi.ac.uk 2023-03-16T11:42:58Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-15T21:38:52Z channel chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:41:31Z ammonium residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:39:03Z conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial structure_element SO: melaniev@ebi.ac.uk 2023-06-14T09:48:13Z segment residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:39:11Z 450–457 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:39:14Z 446–453 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:52Z CaMep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:39:24Z autoinhibitory (AI) region experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T21:39:27Z removal protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:44Z active protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T21:39:30Z transporter protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:39:49Z absence of protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:02Z Npr1 RESULTS paragraph 14939 Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function. structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region protein PR: melaniev@ebi.ac.uk 2023-03-16T08:27:01Z Npr1 site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:26:48Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:26:51Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:31Z TM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:50Z TM4 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:22:36Z main body protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T08:27:09Z transporter structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:28:07Z Val447 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:28:14Z Asp449 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:28:23Z Pro450 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:28:37Z Arg452 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:56Z AI regions protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:53Z CaMep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR site SO: melaniev@ebi.ac.uk 2023-03-16T08:29:21Z Npr1 target serine oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:26:42Z electron density residue_name SO: melaniev@ebi.ac.uk 2023-03-16T11:44:10Z serine protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:07Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:26:54Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:10Z Mep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:11Z inactive protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:26:42Z electron density residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:21Z disordered protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:53Z CaMep2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:33:39Z disorder structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR site SO: melaniev@ebi.ac.uk 2023-03-16T08:29:08Z kinase target site ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:15Z Npr1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:29:14Z disordered structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:33:44Z not conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T08:28:58Z 457Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:31Z truncation mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:33:48Z low activity ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:18Z Npr1 mutant MESH: melaniev@ebi.ac.uk 2023-06-14T09:50:49Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:55Z lacking the His-tag protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-22T09:42:02Z WT site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site RESULTS title_2 16987 Mep2 lacking the AI region is conformationally heterogeneous protein PR: melaniev@ebi.ac.uk 2023-03-16T08:34:05Z Mep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:34:08Z lacking structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:33:59Z conformationally heterogeneous RESULTS paragraph 17048 Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). Why then does this mutant appear to be constitutively active? We propose two possibilities. The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3). residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:41:29Z 453 site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-16T08:41:36Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-16T08:41:39Z channel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:44Z active protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T08:41:46Z transporter ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T08:42:09Z 449-485Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:42:36Z deletion mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:42:13Z lacking structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:41:52Z highly active chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:42:20Z MA mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:27Z triple mepΔ mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:41Z triple mepΔ npr1Δ mutant MESH: melaniev@ebi.ac.uk 2023-03-16T08:42:21Z Mep2 variant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:42:03Z constitutively open site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:36Z channel mutant MESH: melaniev@ebi.ac.uk 2023-03-16T08:42:28Z 446Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:33:56Z mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T08:43:04Z constructed and purified protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:53Z CaMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:40:50Z 442Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:31Z truncation mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T08:42:58Z determined evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:19Z crystal structure evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:22Z structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T08:43:00Z removal of structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:30:32Z cytoplasmic parts protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:30:23Z transporter structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:22:36Z main body protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:23Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:31Z Density structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:25Z Arg415 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T08:43:11Z 442Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:14Z mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:34Z density structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:44Z ICLs structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:28Z structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:15Z Tyr49 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:42Z His342 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bond protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:44Z active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T08:42:55Z crystallization site SO: melaniev@ebi.ac.uk 2023-06-14T09:37:58Z Tyr–His hydrogen bond protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:55Z NH3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T11:42:21Z H+ structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif site SO: melaniev@ebi.ac.uk 2023-06-14T09:38:06Z Tyr–His hydrogen bond experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:41:21Z removal protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:06Z ScMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T08:43:46Z Y53A protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:49Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:43:55Z constitutively active protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T08:44:01Z transporter RESULTS title_2 19155 Phosphorylation causes a conformational change in the CTR ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z Phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR RESULTS paragraph 19213 Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). Overall, ∼20 residues are affected by the introduced mutations. This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to >22 Å after 200 ns simulations, and thus these residues are not interacting. The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to >30 Å after 200 ns. The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. The remainder of the protein is not affected (Supplementary Fig. 5). Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels. protein PR: melaniev@ebi.ac.uk 2023-03-16T09:08:40Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:08:43Z structures ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:00Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:11Z Ser457 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:08:47Z 453 site SO: melaniev@ebi.ac.uk 2023-03-16T09:08:57Z electronegative pocket protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:09:01Z solvent accessible residue_name SO: melaniev@ebi.ac.uk 2023-03-16T11:44:16Z serine residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:02Z Asp419 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:11Z Glu420 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:37Z Glu421 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:54Z determined evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:07:35Z structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:38:37Z phosphorylation-mimicking mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:08:33Z R452D/S453D mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:02:25Z additional mutation of residue_name SO: melaniev@ebi.ac.uk 2023-03-16T09:02:30Z arginine site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T09:09:46Z phosphate structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:22:36Z main body protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:09:59Z transporter chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:42:48Z ammonium species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT protein PR: melaniev@ebi.ac.uk 2023-03-16T09:10:08Z Mep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:08:22Z S453D protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:10:11Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:27Z triple mepΔ structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:10:25Z AI segment protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:10:43Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:07:53Z 12-residue-long α-helix residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:07:58Z Leu427 to Asp438 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:07:49Z Glu420-Leu423 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:37Z Glu421 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:08:12Z disordered protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:30:39Z ammonium transporter experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:08Z mutation ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:01Z R452D experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:13Z determined evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:11:16Z structure mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:22Z single D mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:08:22Z S453D protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:11:29Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:22Z single D experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:32Z mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:37Z DD substitution protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:11:44Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:58:00Z crystal structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:11:52Z modelling experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T16:58:11Z MD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:53Z CaMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:28:47Z phosphorylated mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:12:09Z S453J protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:12:14Z structure residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:02Z Asp419 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:11Z Glu420 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:23:37Z Glu421 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bonding residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T16:58:11Z MD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:41Z simulation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:13:05Z r.m.s.d. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:41Z simulation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:28Z stable protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:28Z stable bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bonds residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:11Z Glu420 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:25Z Ser453 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:11Z Glu420 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:28Z stable experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:52Z simulations evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:14:28Z r.m.s.d protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:51Z WT experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:41Z simulation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:38:34Z Asp452 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:38:54Z Asp453 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:15:34Z distance residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:38:54Z Asp453 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:11Z Glu420 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:52Z simulations protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:15:01Z structurally stable experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:41Z simulation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:12:09Z S453J protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:15:08Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:59:28Z stable experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:41Z simulation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:28:37Z Arg452 residue_name_number DUMMY: means Ser453 melaniev@ebi.ac.uk 2023-03-16T11:40:07Z Sep453 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:41Z simulation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:40:14Z Asp452 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:38:54Z Asp453 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:15:37Z distance chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T09:15:43Z phosphate residue_name_number DUMMY: means Ser453 melaniev@ebi.ac.uk 2023-03-16T11:40:25Z Sep453 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:02:11Z Glu420 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:15:48Z short helix residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:44:49Z Leu427 to Asp438 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:14:52Z simulations protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:34:10Z disordered experimental_method MESH: melaniev@ebi.ac.uk 2023-03-15T16:58:11Z MD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:16:32Z simulations evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:58:00Z crystal structures ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:16:38Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:16:43Z phosphomimetic mutants evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:16:47Z crystals structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR site SO: melaniev@ebi.ac.uk 2023-03-16T09:16:51Z channels protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:16:58Z mutants residue_name SO: melaniev@ebi.ac.uk 2023-03-16T09:17:30Z phosphoserine mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:08:22Z S453D mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:41:10Z DD mutants protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:17:36Z fully active protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:17:49Z absence of protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:33Z Npr1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:17:55Z mutations ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:08:22Z S453D evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:17:59Z structure chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T09:18:29Z ammonium experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:18:06Z crystallization protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:01Z closed protein PR: melaniev@ebi.ac.uk 2023-03-16T09:18:02Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-16T09:18:08Z channels DISCUSS title_1 24519 Discussion DISCUSS paragraph 24530 Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion. protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:44:16Z ammonium transporter evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T09:44:20Z structure taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:24:22Z biochemical and genetic studies taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:43:45Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:24Z eukaryotes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:44:55Z ammonium species MESH: melaniev@ebi.ac.uk 2023-03-16T09:24:37Z Arabidopsis thaliana protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:24:57Z T460 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:44:48Z ammonium protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:05Z transporter protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:01Z phosphomimetic mutations oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:51:45Z ammonium ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs site SO: melaniev@ebi.ac.uk 2023-03-16T09:25:15Z channel taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:19Z transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:11Z inactive protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:45:13Z ammonium protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated site SO: melaniev@ebi.ac.uk 2023-03-16T09:25:37Z channels protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:02Z closed protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:51Z transporters species MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:32Z A. thaliana protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:39Z Mep2 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:34:16Z inactivation taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T17:06:27Z plants protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:34:20Z activation taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:50:14Z fungi taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:50:14Z fungi chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:45:24Z ammonium protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:45:35Z ammonium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:45:45Z ammonium protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:24:10Z Ato protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:46Z transporters DISCUSS paragraph 26215 By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:31:18Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:02Z closed protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:31:21Z comparing evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:31:24Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:31:28Z permanently open taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:46:14Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-22T09:46:31Z channel structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:31:47Z transporters structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 site SO: melaniev@ebi.ac.uk 2023-03-16T09:31:51Z channel structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 site SO: melaniev@ebi.ac.uk 2023-03-22T09:46:57Z channel residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:47Z His2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bonding protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:32:07Z highly conserved residue_name SO: melaniev@ebi.ac.uk 2023-03-16T09:32:10Z tyrosine ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:44Z Npr1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:32:16Z kinase chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:46:44Z nitrogen protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:32:20Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif site SO: melaniev@ebi.ac.uk 2023-03-16T09:32:25Z channel evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:32:28Z structural similarities structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:32:32Z TM parts protein PR: melaniev@ebi.ac.uk 2023-03-16T09:32:35Z Mep2 protein PR: melaniev@ebi.ac.uk 2023-03-16T09:42:52Z AfAmt-1 site SO: melaniev@ebi.ac.uk 2023-03-22T09:47:11Z channel site SO: melaniev@ebi.ac.uk 2023-03-16T09:32:39Z channel site SO: melaniev@ebi.ac.uk 2023-03-22T09:47:52Z channel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer site SO: melaniev@ebi.ac.uk 2023-03-16T09:32:57Z channel structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:43:58Z monomers ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:47:27Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-22T09:47:36Z channel ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation site SO: melaniev@ebi.ac.uk 2023-06-14T09:39:23Z Tyr–His2 interaction chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T09:33:15Z ammonium DISCUSS paragraph 27848 Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins. protein PR: melaniev@ebi.ac.uk 2023-03-16T09:36:43Z Mep2 site SO: melaniev@ebi.ac.uk 2023-03-16T09:36:47Z channels taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:05Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:36:54Z structural data structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:18:42Z cytoplasmic loops protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:02Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:01Z S. cerevisiae species MESH: melaniev@ebi.ac.uk 2023-03-15T16:40:10Z C. albicans evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:37:06Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:37:12Z CTRs structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:39Z AI region structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR site SO: melaniev@ebi.ac.uk 2023-03-16T09:37:19Z Npr1 kinase site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:37:22Z conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:37:27Z transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:53Z absolutely conserved site SO: melaniev@ebi.ac.uk 2023-06-14T09:39:36Z ICL1-ICL3-CTR interaction network taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:48:32Z ammonium protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:37:39Z conserved protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:37:42Z Mep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:50:14Z fungi chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:48:41Z ammonium species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:02Z S. cerevisiae residue_name SO: melaniev@ebi.ac.uk 2023-03-16T09:37:49Z tyrosine residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:47Z His2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:37:54Z highly conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:37:59Z Mep2 site SO: melaniev@ebi.ac.uk 2023-06-14T09:39:47Z Tyr–His2 hydrogen bond protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:34:30Z close protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins DISCUSS paragraph 29070 With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:24:57Z T460 site SO: melaniev@ebi.ac.uk 2023-03-16T09:42:14Z pore structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:42:36Z C terminus experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:42:40Z homology model protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:52:15Z archaebacterial protein PR: melaniev@ebi.ac.uk 2023-03-16T09:42:52Z AfAmt-1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:42:55Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:43:00Z C terminus protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer protein PR: melaniev@ebi.ac.uk 2023-03-16T09:43:06Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:43:09Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:24:57Z T460 protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:43:14Z structural information protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:06Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:26Z Y467 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:35Z H239 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:42Z D458 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:27:50Z K71 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:53Z CaMep2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:15Z Tyr49 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs site SO: melaniev@ebi.ac.uk 2023-06-14T09:40:12Z Tyr–His2 hydrogen bond protein PR: melaniev@ebi.ac.uk 2023-03-16T09:43:43Z Mep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:02Z closed taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:43:45Z transporters DISCUSS paragraph 30177 We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2. site SO: melaniev@ebi.ac.uk 2023-06-14T09:40:25Z intra-monomeric CTR-ICL1/ICL3 interactions taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open site SO: melaniev@ebi.ac.uk 2023-03-16T09:48:17Z channels protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:48:13Z lack of protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:11Z inactive site SO: melaniev@ebi.ac.uk 2023-03-15T21:38:02Z phosphorylation sites structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs protein PR: melaniev@ebi.ac.uk 2023-03-16T09:49:31Z Mep2 ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation site SO: melaniev@ebi.ac.uk 2023-03-22T09:49:17Z channel protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs site SO: melaniev@ebi.ac.uk 2023-03-22T09:49:28Z channel protein PR: melaniev@ebi.ac.uk 2023-03-16T09:49:51Z Mep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:39Z certain mutations structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR residue_name SO: melaniev@ebi.ac.uk 2023-03-16T11:44:21Z glycine structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:56Z Mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:50:01Z G393 protein PR: melaniev@ebi.ac.uk 2023-03-16T09:50:05Z EcAmtB residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T09:50:08Z G456 protein PR: melaniev@ebi.ac.uk 2023-03-15T21:27:13Z AtAmt-1;1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:50:13Z transporters species MESH: melaniev@ebi.ac.uk 2023-03-16T09:50:17Z Escherichia coli protein PR: melaniev@ebi.ac.uk 2023-03-16T09:50:21Z AmtB species MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:32Z A. thaliana protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:02Z closed protein PR: melaniev@ebi.ac.uk 2023-03-16T09:50:31Z Mep2 DISCUSS paragraph 31335 Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. Nevertheless, as discussed above, considerable differences may exist between different species. ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T10:57:58Z aquaporins protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T10:58:00Z urea transporters taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:05Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T10:58:06Z channels protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T10:58:09Z transporters ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T10:58:14Z nitrate transporters chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:49:43Z ammonium ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation species MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:32Z A. thaliana protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:02Z S. cerevisiae protein PR: melaniev@ebi.ac.uk 2023-03-16T11:01:16Z Mep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:01:05Z absence of protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:30:49Z GlnK proteins taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:24Z eukaryotes ptm MESH: melaniev@ebi.ac.uk 2023-03-22T09:50:00Z phosphorylation chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:50:14Z ammonium DISCUSS paragraph 32018 With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations. protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T09:50:30Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters protein PR: melaniev@ebi.ac.uk 2023-03-16T11:02:50Z Mep1 protein PR: melaniev@ebi.ac.uk 2023-03-15T16:52:05Z Mep3 protein PR: melaniev@ebi.ac.uk 2023-03-16T11:02:56Z Mep2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:55Z NH3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-15T16:58:55Z NH3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T11:03:06Z H+ chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T11:03:03Z NH4+ protein PR: melaniev@ebi.ac.uk 2023-03-16T11:03:10Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:03:12Z transporter evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:03:14Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:42:15Z Mep2 proteins DISCUSS paragraph 32878 It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate. Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:50:51Z ammonium protein PR: melaniev@ebi.ac.uk 2023-03-16T11:03:44Z Mep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:31:05Z eukaryotic chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:51:02Z ammonium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:51:15Z ammonium taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:50:14Z fungi METHODS title_1 33364 Methods METHODS title_2 33372 Mep2 overexpression and purification METHODS paragraph 33409 Ammonium transporter genes were amplified from genomic DNA or cDNA by PCR (Phusion, New England Biolabs). In both ScMEP2 and CaMEP2, Asn4 was replaced by a glutamine to prevent glycosylation. In order to allow transformation of yeast by recombination, the following primer extensions were used: forward 5′-GAAAAAACCCCGGATTCTAGAACTAGTGGATCCTCC-3′ and reverse 5′-TGACTCGAGTTATGCACCGTGGTGGTGATGGTGATG-3′. These primers result in a construct that lacks the cleavable N- and C-terminal tags present in the original vector, and replaces these with a C-terminal hexa-histidine tag. Recombination in yeast strain W303 pep4Δ was carried out using ∼50–100 ng of SmaI-digested vector 83νΔ (ref.) and at least a fourfold molar excess of PCR product via the lithium acetate method. Transformants were selected on SCD -His plates incubated at 30 °C. Construction of mutant CaMEP2 genes was done using the Q5 site-directed mutagenesis kit (NEB) per manufacturer's instructions. Three CaMep2 mutants were made for crystallization: the first mutant is a C-terminal truncation mutant 442Δ, lacking residues 443–480 including the AI domain. The second mutant, R452D/S453D, mimics the protein phosphorylated at Ser453. Given that phosphate is predominantly charged −2 at physiological pH, we introduced the second aspartate residue for Arg452. However, we also constructed the ‘single D', S453D CaMep2 variant. METHODS paragraph 34827 For expression, cells were grown in shaker flasks at 30 °C for ∼24 h in synthetic minimal medium lacking histidine and with 1% (w/v) glucose to a typical OD600 of 6–8. Cells were subsequently spun down for 15 min at 4,000g and resuspended in YP medium containing 1.5% (w/v) galactose, followed by another 16–20 h growth at 30 °C/160 r.p.m. and harvesting by centrifugation. Final OD600 values typically reached 18–20. Cells were lysed by bead beating (Biospec) for 5 × 1 min with 1 min intervals on ice, or by 1–2 passes through a cell disrupter operated at 35,000 p.s.i. (TS-Series 0.75 kW; Constant Systems). Membranes were collected from the suspension by centrifugation at 200,000g for 90 min (45Ti rotor; Beckmann Coulter). Membrane protein extraction was performed by homogenization in a 1:1 (w/w) mixture of dodecyl-β-D-maltoside and decyl-β-D-maltoside (DDM/DM) followed by stirring at 4 °C overnight. Typically, 1 g (1% w/v) of total detergent was used for membranes from 2 l of cells. The membrane extract was centrifuged for 35 min at 200,000g and the supernatant was loaded onto a 10-ml Nickel column (Chelating Sepharose; GE Healthcare) equilibrated in 20 mM Tris/300 mM NaCl/0.2% DDM, pH 8. The column was washed with 15 column volumes buffer containing 30 mM imidazole and eluted in 3 column volumes with 250 mM imidazole. Proteins were purified to homogeneity by gel filtration chromatography in 10 mM HEPES/100 mM NaCl/0.05% DDM, pH 7–7.5. For polishing and detergent exchange, a second gel filtration column was performed using various detergents. In the case of ScMep2, diffracting crystals were obtained only with 0.05% decyl-maltose neopentyl glycol. For the more stable CaMep2 protein, we obtained crystals in, for example, nonyl-glucoside, decyl-maltoside and octyl-glucose neopentyl glycol. Proteins were concentrated to 7–15 mg ml−1 using 100 kDa cutoff centrifugal devices (Millipore), flash-frozen and stored at −80 °C before use. METHODS title_2 36860 Crystallization and structure determination METHODS paragraph 36904 Crystallization screening trials by sitting drop vapour diffusion were set up at 4 and 20 °C using in-house screens and the MemGold 1 and 2 screens (Molecular Dimensions) with a Mosquito crystallization robot. Crystals were harvested directly from the initial trials or optimized by sitting or hanging drop vapour diffusion using larger drops (typically 2–3 μl total volume). Bar-shaped crystals for ScMep2 diffracting to 3.2 Å resolution were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)/0.2 M di-ammonium hydrogen phosphate/30% PEG 400, pH 6. They belong to space group P212121 and have nine molecules (three trimers) in the asymmetric unit (AU). Well-diffracting crystals for CaMep2 were obtained in space group P3 from 0.1 M MES/0.2 M lithium sulphate/20% PEG400, pH 6 (two molecules per AU). An additional crystal form in space group R3 was grown in 0.04 M Tris/0.04 M NaCl/27% PEG350 MME, pH 8 (one molecule per AU). Diffracting crystals for the phosporylation-mimicking CaMep2 DD mutant were obtained in space group P6322 from 0.1 M sodium acetate/15–20% PEG400, pH 5 (using decyl-maltoside as detergent; one molecule per AU), while S453D mutant crystals grew in 24% PEG400/0.05 M sodium acetate, pH 5.4/0.05 M magnesium acetate tetrahydrate/10 mM NH4Cl (space group R32; one molecule per AU). Finally, the 442Δ truncation mutant gave crystals under many different conditions, but most of these diffracted poorly or not at all. A reasonable low-resolution data set (3.4 Å resolution) was eventually obtained from a crystal grown in 24% PEG400/0.05 M sodium acetate/0.05 M magnesium acetate, pH 6.1 (space group R32). Diffraction data were collected at the Diamond Light Source and processed with XDS or HKL2000 (ref. ). METHODS paragraph 38689 For MR, a search model was constructed with Sculptor within Phenix, using a sequence alignment of ScMep2 with Archaeoglobus fulgidus Amt-1 (PDB ID 2B2H; ∼40% sequence identity to ScMep2). A clear solution with nine molecules (three trimers) in the AU was obtained using Phaser. The model was subsequently completed by iterative rounds of manual building within Coot followed by refinement within Phenix. The structures for WT CaMep2 were solved using the best-defined monomer of ScMep2 (60% sequence identity with CaMep2) in MR with Phaser, followed by automated model building within Phenix. Finally, the structures of the three mutant CaMep2 proteins were solved using WT CaMep2 as the search model. The data collection and refinement statistics for all six solved structures have been summarized in Supplementary Tables 1 and 2. METHODS title_2 39523 Growth assays METHODS paragraph 39537 The S. cerevisiae haploid triple mepΔ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 ura3-52) and triple mepΔ npr1Δ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 npr1::NAT1 ura3-52) were generated by integrating the NAT1 resistance gene at one NPR1 locus in the diploid strain MLY131 (ref.), followed by isolation of individual haploid strains. Cells were grown in synthetic minimal medium with glucose (2%) as the carbon source and ammonium sulphate (1 mM) or glutamate (0.1%) as the nitrogen source. Yeast cells were transformed as described. All DNA sequences encoding epitope-tagged ScMep2 and its mutant derivatives were generated by PCR and homologous recombination using the vector pRS316 (ref. ). In each case, the ScMEP2 sequences included the ScMEP2 promoter (1 kb), the ScMEP2 terminator and sequences coding for a His-6 epitope at the C-terminal end of the protein. All Mep2-His fusions contain the N4Q mutation to prevent glycosylation of Mep2 (ref.). All newly generated plasmid inserts were verified by DNA sequencing. For growth assays, S. cerevisiae cells containing plasmids expressing ScMep2 or mutant derivatives were grown overnight in synthetic minimal glutamate medium, washed, spotted by robot onto solid agar plates and culture growth followed by time course photography. Images were then processed to quantify the growth of each strain over 3 days as described. METHODS title_2 40966 Protein modelling METHODS paragraph 40984 The MODELLER (version 9.15) software package was used to build protein structures for MD simulations. This method was required to construct two complete protein models, the double mutant R452D/S453D (with the four missing residues from the X-ray structure added) and also the construct in which the mutation at position 452 is reverted to R, and D453 is replaced with a phosphoserine. The quality of these models was assessed using normalized Discrete Optimized Protein Energy (DOPE) values and the molpdf assessment function within the MODELLER package. The model R452D/S453D mutant has a molpdf assessment score of 1854.05, and a DOPE assessment score of -60920.55. The model of the S453J mutant has a molpdf assessment score of 1857.01 and a DOPE assessment score of −61032.15. METHODS title_2 41767 MD simulations METHODS paragraph 41782 WT and model structures were embedded into a pre-equilibrated lipid bilayer composed of 512 dipalmitoylphosphatidylcholine lipids using the InflateGRO2 computer programme. The bilayers were then solvated with the SPC water model and counterions were added to achieve a charge neutral state. All simulations were performed with the GROMACS package (version 4.5.5), and the GROMOS96 43a1p force field. During simulation time, the temperature was maintained at 310 K using the Nosé-Hoover thermostat with a coupling constant of 0.5 ps. Pressure was maintained at 1 bar using semi-isotropic coupling with the Parrinello-Rahman barostat and a time constant of 5 ps. Electrostatic interactions were treated using the smooth particle mesh Ewald algorithm with a short-range cutoff of 0.9 nm. Van der Waals interactions were truncated at 1.4 nm with a long-range dispersion correction applied to energy and pressure. The neighbour list was updated every five steps. All bonds were constrained with the LINCS algorithm, so that a 2-fs time step could be applied throughout. The phospholipid parameters for the dipalmitoylphosphatidylcholine lipids were based on the work of Berger. The embedded proteins were simulated for 200 ns each; a repeat simulation was performed for each system with different initial velocities to ensure reproducibility. To keep the c.p.u. times within reasonable limits, all simulations were performed on Mep2 monomers. This is also consistent with previous simulations for E. coli AmtB. METHODS title_1 43303 Additional information METHODS paragraph 43326 Accession codes: The atomic coordinates and the associated structure factors have been deposited in the Protein Data Bank (http:// www.pdbe.org) with accession codes 5AEX (ScMep2), 5AEZ(CaMep2; R3), 5AF1(CaMep2; P3), 5AID(CaMep2; 442D), 5AH3 (CaMep2; R452D/S453D) and 5FUF (CaMep2; S453D). METHODS paragraph 43616 How to cite this article: van den Berg, B. et al. Structural basis for Mep2 ammonium transceptor activation by phosphorylation. Nat. Commun. 7:11337 doi: 10.1038/ncomms11337 (2016). SUPPL title_1 43798 Supplementary Material 556 564 surname:Holsbeeks;given-names:I. surname:Lagatie;given-names:O. surname:Van Nuland;given-names:A. surname:Van de Velde;given-names:S. surname:Thevelein;given-names:J. M. 15450611 REF Trends Biochem. Sci. ref 29 2004 43821 The eukaryotic plasma membrane as a nutrient-sensing device 254 299 surname:Conrad;given-names:M. 24483210 REF FEMS Microbiol. 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Chem. ref 26 2005 48839 GROMACS: fast, flexible, and free 511 519 surname:Nosé;given-names:S. REF J. Chem. Phys. ref 81 1984 48873 A unified formulation of the constant temperature molecular dynamics methods 1695 surname:Hoover;given-names:W. G. REF Phys. Rev. A ref 31 1985 48950 Canonical dynamics: equilibrium phase-space distributions 7182 7190 surname:Parrinello;given-names:M. surname:Rahman;given-names:A. REF J. Appl. Phys. ref 52 1981 49008 Polymorphic transitions in single crystals: a new molecular dynamics method 8577 8593 surname:Essmann;given-names:U. REF J. Chem. Phys. ref 103 1995 49084 A smooth particle mesh Ewald method 1463 1472 surname:Hess;given-names:B. surname:Bekker;given-names:H. surname:Berendsen;given-names:H. J. surname:Fraaije;given-names:J. G. REF J. Comput. Chem. ref 18 1997 49120 LINCS: a linear constraint solver for molecular simulations 2002 2013 surname:Berger;given-names:O. surname:Edholm;given-names:O. surname:Jähnig;given-names:F. 9129804 REF Biophys. J. ref 72 1997 49180 Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature REF ref 49327 The PyMOL Molecular Graphics System. version 1.7.4 (Schrödinger, LLC). SUPPL footnote 49400 Author contributions B.v.d.B. performed the experiments related to Mep2 structure determination, designed research and wrote the paper. A.C. performed ammonium growth experiments of Mep variants. D.J. and S.K. performed modelling studies and MD simulations. A.B. collected the X-ray synchrotron data and maintained the Newcastle Structural Biology Laboratory. J.C.R. designed research related to the S. cerevisiae growth assays. ncomms11337-f1.jpg f1 FIG fig_title_caption 49829 X-ray crystal structures of Mep2 transceptors. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:22Z X-ray crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-16T11:07:14Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:44:36Z transceptors ncomms11337-f1.jpg f1 FIG fig_caption 49876 (a) Monomer cartoon models viewed from the side for (left) A. fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and C. albicans Mep2 (right). The cartoons are in rainbow representation. The region showing ICL1 (blue), ICL3 (green) and the CTR (red) is boxed for comparison. (b) CaMep2 trimer viewed from the intracellular side (right). One monomer is coloured as in a and one monomer is coloured by B-factor (blue, low; red; high). The CTR is boxed. (c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating the differences in the CTRs. All structure figures were generated with Pymol. oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z Monomer species MESH: melaniev@ebi.ac.uk 2023-03-16T11:08:55Z A. fulgidus protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:24Z Amt-1 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:50:02Z S. cerevisiae protein PR: melaniev@ebi.ac.uk 2023-03-16T11:43:49Z Mep2 species MESH: melaniev@ebi.ac.uk 2023-03-15T16:40:10Z C. albicans protein PR: melaniev@ebi.ac.uk 2023-03-16T11:09:00Z Mep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:41:29Z Overlay protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:07Z ScMep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:37:12Z CTRs ncomms11337-f2.jpg f2 FIG fig_title_caption 50476 Sequence conservation in ammonium transporters. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:09:27Z Sequence conservation protein_type MESH: melaniev@ebi.ac.uk 2023-03-15T16:41:48Z ammonium transporters ncomms11337-f2.jpg f2 FIG fig_caption 50524 ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E. coli AmtB and A. thaliana Amt-1;1. The secondary structure elements observed for CaMep2 are indicated, with the numbers corresponding to the centre of the TM segment. Important regions are labelled. The conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan, the conserved ExxGxD motif of the CTR in red and the AI region in yellow. Coloured residues are functionally important and correspond to those of the Phe gate (blue), the binding site Trp residue (magenta) and the twin-His motif (red). The Npr1 kinase site in the AI region is highlighted pink. The grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the structures and are likely disordered. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:41:36Z ClustalW alignment protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:07Z ScMep2 species MESH: melaniev@ebi.ac.uk 2023-03-16T11:11:32Z A. fulgidus protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:24Z Amt-1 species MESH: melaniev@ebi.ac.uk 2023-03-16T11:11:38Z E. coli protein PR: melaniev@ebi.ac.uk 2023-03-16T11:11:41Z AmtB species MESH: melaniev@ebi.ac.uk 2023-03-16T09:25:32Z A. thaliana protein PR: melaniev@ebi.ac.uk 2023-03-16T09:24:49Z Amt-1;1 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:12:14Z TM segment protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:11:44Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:06:51Z RxK motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:11:48Z ER motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:13:11Z ICL2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:11:51Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:40Z AI region site SO: melaniev@ebi.ac.uk 2023-03-15T18:56:01Z Phe gate site SO: melaniev@ebi.ac.uk 2023-03-16T11:11:57Z binding site residue_name SO: melaniev@ebi.ac.uk 2023-03-16T11:11:54Z Trp structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:57Z twin-His motif site SO: melaniev@ebi.ac.uk 2023-03-16T11:12:01Z Npr1 kinase site structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:40Z AI region protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:07Z ScMep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:12:04Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:12:08Z likely disordered ncomms11337-f3.jpg f3 FIG fig_title_caption 51276 Growth of ScMep2 variants on low ammonium medium. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:14:22Z Growth mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:13:11Z ScMep2 variants ncomms11337-f3.jpg f3 FIG fig_caption 51326 (a) The triple mepΔ strain (black) and triple mepΔ npr1Δ strain (grey) containing plasmids expressing WT and variant ScMep2 were grown on minimal medium containing 1 mM ammonium sulphate. The quantified cell density reflects logarithmic growth after 24 h. Error bars are the s.d. for three replicates of each strain (b) The strains used in a were also serially diluted and spotted onto minimal agar plates containing glutamate (0.1%) or ammonium sulphate (1 mM), and grown for 3 days at 30 °C. mutant MESH: melaniev@ebi.ac.uk 2023-03-15T21:37:27Z triple mepΔ mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:14:11Z triple mepΔ npr1Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:52Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:14:15Z variant ScMep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:14:19Z grown on minimal medium chemical CHEBI: melaniev@ebi.ac.uk 2023-03-16T11:14:25Z ammonium sulphate evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:14:28Z cell density chemical CHEBI: melaniev@ebi.ac.uk 2023-06-15T15:03:58Z glutamate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T09:52:07Z ammonium sulphate ncomms11337-f4.jpg f4 FIG fig_title_caption 51832 Structural differences between Mep2 and bacterial ammonium transporters. protein PR: melaniev@ebi.ac.uk 2023-03-16T11:14:51Z Mep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:41:28Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:14:55Z ammonium transporters ncomms11337-f4.jpg f4 FIG fig_caption 51905 (a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing unwinding and inward movement in the fungal protein. (b) Stereo diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in AfAmt-1 (light colours) and CaMep2 (dark colours). The side chains of residues in the RxK motif as well as those of Tyr49 and His342 are labelled. The numbering is for CaMep2. (c) Conserved residues in ICL1-3 and the CTR. Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the large differences in conformation of the conserved residues in ICL1 (RxK motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). The labelled residues are analogous within both structures. In b and c, the centre of the trimer is at top. structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 protein PR: melaniev@ebi.ac.uk 2023-03-16T09:42:52Z AfAmt-1 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:36:02Z fungal structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR protein PR: melaniev@ebi.ac.uk 2023-03-16T09:42:52Z AfAmt-1 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:06:51Z RxK motif residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:15Z Tyr49 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:42Z His342 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:16:55Z Conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:36:09Z ICL1-3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 protein PR: melaniev@ebi.ac.uk 2023-03-16T09:42:52Z AfAmt-1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:17:09Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:04Z ICL1 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:36:16Z RxK motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T21:13:11Z ICL2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:17:06Z ER motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:17:12Z structures oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer ncomms11337-f5.jpg f5 FIG fig_title_caption 52652 Channel closures in Mep2. protein PR: melaniev@ebi.ac.uk 2023-03-16T11:17:25Z Mep2 ncomms11337-f5.jpg f5 FIG fig_caption 52678 (a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1 that forms a hydrogen bond with His2 in CaMep2. (b) Surface views from the side in rainbow colouring, showing the two-tier channel block (indicated by the arrows) in CaMep2. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:18:26Z superposition protein PR: melaniev@ebi.ac.uk 2023-03-16T09:42:52Z AfAmt-1 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 site SO: melaniev@ebi.ac.uk 2023-03-15T18:56:01Z Phe gate residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:47Z His2 structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:37:38Z twin-His motif residue_name SO: melaniev@ebi.ac.uk 2023-03-16T11:18:29Z tyrosine residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:40:31Z Y49 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:34:46Z TM1 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:21:51Z hydrogen bond residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:47Z His2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T11:35:06Z channel block protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 ncomms11337-f6.jpg f6 FIG fig_title_caption 53000 The Npr1 kinase target Ser453 is dephosphorylated and located in an electronegative pocket. protein PR: melaniev@ebi.ac.uk 2023-03-16T11:19:02Z Npr1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:19:05Z kinase residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:26Z Ser453 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:19:07Z dephosphorylated site SO: melaniev@ebi.ac.uk 2023-03-16T09:08:57Z electronegative pocket ncomms11337-f6.jpg f6 FIG fig_caption 53092 (a) Stereoviews of CaMep2 showing 2Fo–Fc electron density (contoured at 1.0 σ) for CTR residues Asp419-Met422 and for Tyr446-Thr455 of the AI region. For clarity, the residues shown are coloured white, with oxygen atoms in red and nitrogen atoms in blue. The phosphorylation target residue Ser453 is labelled in bold. (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green), showing the similar electronegative environment surrounding the phosphorylation site (P). The AI regions are coloured magenta. (c) Cytoplasmic view of the Mep2 trimer indicating the large distance between Ser453 and the channel exits (circles; Ile52 lining the channel exit is shown). protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:20:42Z 2Fo–Fc electron density structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:20:49Z Asp419-Met422 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:20:52Z Tyr446-Thr455 structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:40Z AI region ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:26Z Ser453 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:21:09Z Overlay structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:37:12Z CTRs protein PR: melaniev@ebi.ac.uk 2023-03-15T18:51:08Z ScMep2 protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:56Z AI regions protein PR: melaniev@ebi.ac.uk 2023-03-16T11:21:18Z Mep2 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:35:26Z Ser453 site SO: melaniev@ebi.ac.uk 2023-03-16T11:21:23Z channel exits residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:21:25Z Ile52 site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit ncomms11337-f7.jpg f7 FIG fig_title_caption 53761 Effect of removal of the AI region on Mep2 structure. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:21:55Z removal structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:40Z AI region protein PR: melaniev@ebi.ac.uk 2023-03-16T11:21:58Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:22:00Z structure ncomms11337-f7.jpg f7 FIG fig_caption 53815 (a) Side views for WT CaMep2 (left) and the truncation mutant 442Δ (right). The latter is shown as a putty model according to B-factors to illustrate the disorder in the protein on the cytoplasmic side. Missing regions are labelled. (b) Stereo superpositions of WT CaMep2 and the truncation mutant. 2Fo–Fc electron density (contoured at 1.0 σ) for residues Tyr49 and His342 is shown for the truncation mutant. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:52Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:31Z truncation mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:22:48Z 442Δ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:22:51Z disorder experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:22:54Z superpositions protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:52Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:31Z truncation mutant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:22:58Z 2Fo–Fc electron density residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:15Z Tyr49 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-15T21:04:42Z His342 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T08:27:31Z truncation mutant ncomms11337-f8.jpg f8 FIG fig_title_caption 54233 Phosphorylation causes conformational changes in the CTR. ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:01Z Phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR ncomms11337-f8.jpg f8 FIG fig_caption 54291 (a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2 superposed in grey for one of the monomers. The arrow indicates the phosphorylation site. The AI region is coloured magenta. (b) Monomer side-view superposition of WT CaMep2 and the DD mutant, showing the conformational change and disorder around the ExxGxD motif. Side chains for residues 452 and 453 are shown as stick models. mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:52Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:24:27Z superposed oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:44:04Z monomers site SO: melaniev@ebi.ac.uk 2023-03-15T16:38:22Z phosphorylation site structure_element SO: melaniev@ebi.ac.uk 2023-03-16T08:25:40Z AI region oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z Monomer experimental_method MESH: melaniev@ebi.ac.uk 2023-03-16T11:24:36Z superposition protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:02:52Z WT protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:01:49Z DD mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:24:40Z 452 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:24:42Z 453 ncomms11337-f9.jpg f9 FIG fig_title_caption 54681 Schematic model for phosphorylation-based regulation of Mep2 ammonium transporters. protein PR: melaniev@ebi.ac.uk 2023-03-16T11:25:03Z Mep2 protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:25:06Z ammonium transporters ncomms11337-f9.jpg f9 FIG fig_caption 54765 (a) In the closed, non-phosphorylated state (i), the CTR (magenta) and ICL3 (green) are far apart with the latter blocking the intracellular channel exit (indicated with a hatched circle). Upon phosphorylation and mimicked by the CaMep2 S453D and DD mutants (ii), the region around the ExxGxD motif undergoes a conformational change that results in the CTR interacting with the inward-moving ICL3, opening the channel (full circle) (iii). The arrows depict the movements of important structural elements. The open-channel Mep2 structure is represented by archaebacterial Amt-1 and shown in lighter colours consistent with Fig. 4. As discussed in the text, similar structural arrangements may occur in plant AMTs. In this case however, the open channel corresponds to the non-phosphorylated state; phosphorylation breaks the CTR–ICL3 interactions leading to channel closure. (b) Model based on AMT transporter analogy showing how phosphorylation of a Mep2 monomer might allosterically open channels in the entire trimer via disruption of the interactions between the CTR and ICL3 of a neighbouring monomer (arrow). protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:02Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:46Z channel exit ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:02Z phosphorylation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:27:54Z mimicked protein PR: melaniev@ebi.ac.uk 2023-03-15T18:50:54Z CaMep2 mutant MESH: melaniev@ebi.ac.uk 2023-03-16T09:08:22Z S453D mutant MESH: melaniev@ebi.ac.uk 2023-03-16T11:41:10Z DD mutants structure_element SO: melaniev@ebi.ac.uk 2023-03-16T09:08:08Z ExxGxD motif structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 site SO: melaniev@ebi.ac.uk 2023-03-16T11:37:51Z channel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open site SO: melaniev@ebi.ac.uk 2023-03-16T11:28:01Z channel protein PR: melaniev@ebi.ac.uk 2023-03-16T11:28:04Z Mep2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:28:07Z structure taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:52:15Z archaebacterial protein PR: melaniev@ebi.ac.uk 2023-03-15T18:52:24Z Amt-1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-15T19:03:15Z plant protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T09:49:46Z AMTs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open site SO: melaniev@ebi.ac.uk 2023-03-16T11:28:11Z channel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:39:42Z non-phosphorylated ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:02Z phosphorylation site SO: melaniev@ebi.ac.uk 2023-06-14T09:41:01Z CTR–ICL3 interactions site SO: melaniev@ebi.ac.uk 2023-03-22T09:53:12Z channel protein_type MESH: melaniev@ebi.ac.uk 2023-03-16T11:28:14Z AMT transporter ptm MESH: melaniev@ebi.ac.uk 2023-03-15T17:14:02Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-16T11:28:22Z Mep2 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T16:37:21Z open site SO: melaniev@ebi.ac.uk 2023-03-22T09:53:22Z channels oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-16T11:08:50Z trimer structure_element SO: melaniev@ebi.ac.uk 2023-03-15T16:38:07Z CTR structure_element SO: melaniev@ebi.ac.uk 2023-03-15T19:01:14Z ICL3 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-15T18:50:28Z monomer