PMC 20140719 pmc.key 4833862 CC BY no 0 0 10.1038/ncomms11196 ncomms11196 4833862 27073141 11196 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:Hunkeler;given-names:Moritz surname:Stuttfeld;given-names:Edward surname:Hagmann;given-names:Anna surname:Imseng;given-names:Stefan surname:Maier;given-names:Timm TITLE front 7 2016 0 The dynamic organization of fungal acetyl-CoA carboxylase protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:28Z dynamic taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:48:20Z acetyl-CoA carboxylase ABSTRACT abstract 58 Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:58:07Z Acetyl-CoA carboxylases protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:22:38Z ATP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:51:14Z acetyl-CoA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:51:34Z malonyl-CoA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z Eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:51Z single-chain multienzymes protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:51:54Z non-catalytic structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:51:56Z central domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:44Z ACC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated site SO: melaniev@ebi.ac.uk 2023-03-17T16:53:27Z phosphorylation site taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:04Z structure mutant MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:10Z larger fragments protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:17Z intact protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:28Z dynamic taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:56:12Z carboxylases structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation ABSTRACT abstract 1127 Acetyl-CoA carboxylases are central regulatory hubs of fatty acid metabolism and are important targets for drug development in obesity and cancer. Here, the authors demonstrate that the regulation of these highly dynamic enzymes in fungi is governed by a mechanism based on phosphorylation-dependent conformational variability. protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:58:07Z Acetyl-CoA carboxylases protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:58:13Z highly dynamic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:58:16Z enzymes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:58:19Z fungi ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation INTRO paragraph 1456 Biotin-dependent acetyl-CoA carboxylases (ACCs) are essential enzymes that catalyse the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. This reaction provides the committed activated substrate for the biosynthesis of fatty acids via fatty-acid synthase. By catalysing this rate-limiting step in fatty-acid biosynthesis, ACC plays a key role in anabolic metabolism. ACC inhibition and knock-out studies show the potential of targeting ACC for treatment of the metabolic syndrome. Furthermore, elevated ACC activity is observed in malignant tumours. A direct link between ACC and cancer is provided by cancer-associated mutations in the breast cancer susceptibility gene 1 (BRCA1), which relieve inhibitory interactions of BRCA1 with ACC. Thus, ACC is a relevant drug target for type 2 diabetes and cancer. Microbial ACCs are also the principal target of antifungal and antibiotic compounds, such as Soraphen A. protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:52:09Z Biotin-dependent acetyl-CoA carboxylases protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:22:53Z ATP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:51:14Z acetyl-CoA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:51:34Z malonyl-CoA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:23:23Z fatty acids protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:00:40Z fatty-acid synthase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:00:49Z ACC inhibition and knock-out studies protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:23:04Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC mutant MESH: melaniev@ebi.ac.uk 2023-03-17T17:01:06Z mutations protein PR: melaniev@ebi.ac.uk 2023-03-17T17:01:02Z breast cancer susceptibility gene 1 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:01:12Z BRCA1 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:01:12Z BRCA1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:01:23Z Microbial protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T17:01:17Z Soraphen A INTRO paragraph 2376 The principal functional protein components of ACCs have been described already in the late 1960s for Escherichia coli (E. coli) ACC: Biotin carboxylase (BC) catalyses the ATP-dependent carboxylation of a biotin moiety, which is covalently linked to the biotin carboxyl carrier protein (BCCP). Carboxyltransferase (CT) transfers the activated carboxyl group from carboxybiotin to acetyl-CoA to yield malonyl-CoA. Prokaryotic ACCs are transient assemblies of individual BC, CT and BCCP subunits. Eukaryotic ACCs, instead, are multienzymes, which integrate all functional components into a single polypeptide chain of ∼2,300 amino acids. Human ACC occurs in two closely related isoforms, ACC1 and 2, located in the cytosol and at the outer mitochondrial membrane, respectively. In addition to the canonical ACC components, eukaryotic ACCs contain two non-catalytic regions, the large central domain (CD) and the BC–CT interaction domain (BT). The CD comprises one-third of the protein and is a unique feature of eukaryotic ACCs without homologues in other proteins. The function of this domain remains poorly characterized, although phosphorylation of several serine residues in the CD regulates ACC activity. The BT domain has been visualized in bacterial carboxylases, where it mediates contacts between α- and β-subunits. protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs species MESH: melaniev@ebi.ac.uk 2023-03-17T17:04:36Z Escherichia coli species MESH: melaniev@ebi.ac.uk 2023-03-17T17:04:43Z E. coli protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:04:49Z Biotin carboxylase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:04:52Z BC chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:23:40Z ATP chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T17:04:55Z biotin protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:00Z biotin carboxyl carrier protein protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:03Z BCCP protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:06Z Carboxyltransferase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:09Z CT chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T17:05:16Z carboxyl chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T17:05:19Z carboxybiotin chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:51:14Z acetyl-CoA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T16:51:34Z malonyl-CoA taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:32Z Prokaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:13:06Z transient protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:43Z BC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:46Z CT protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:49Z BCCP taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z Eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:05:59Z multienzymes species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z Human protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:06:46Z isoforms protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:06:52Z 2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:03Z ACC components taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:07:12Z non-catalytic structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:16Z regions structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:20Z central domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:30Z BC–CT interaction domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:37Z BT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:07:43Z unique feature of taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name SO: melaniev@ebi.ac.uk 2023-03-17T17:08:22Z serine structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:23:56Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:37Z BT taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:08:41Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:56:12Z carboxylases structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:08:31Z α- structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:08:44Z β-subunits INTRO paragraph 3706 Structural studies on the functional architecture of intact ACCs have been hindered by their huge size and pronounced dynamics, as well as the transient assembly mode of bacterial ACCs. However, crystal structures of individual components or domains from prokaryotic and eukaryotic ACCs, respectively, have been solved. The structure determination of the holoenzymes of bacterial biotin-dependent carboxylases, which lack the characteristic CD, such as the pyruvate carboxylase (PC), propionyl-CoA carboxylase, 3-methyl-crotonyl-CoA carboxylase and a long-chain acyl-CoA carboxylase revealed strikingly divergent architectures despite a general conservation of all functional components. In these structures, the BC and CT active sites are at distances between 40 and 80 Å, such that substrate transfer could be mediated solely by the mobility of the flexibly tethered BCCP. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:12:57Z Structural studies protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:17Z intact protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:13:06Z transient taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:08:41Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:18Z crystal structures taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:32Z prokaryotic taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:13:21Z structure determination protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:13:24Z holoenzymes taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:08:41Z bacterial protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:13:28Z biotin-dependent carboxylases protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:13:58Z lack structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:13:47Z pyruvate carboxylase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:02Z PC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:05Z propionyl-CoA carboxylase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:08Z 3-methyl-crotonyl-CoA carboxylase protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:14Z long-chain acyl-CoA carboxylase evidence DUMMY: melaniev@ebi.ac.uk 2023-06-15T14:33:06Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:21Z BC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:24Z CT site SO: melaniev@ebi.ac.uk 2023-03-17T17:14:31Z active sites protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:14:37Z flexibly tethered protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T17:14:42Z BCCP INTRO paragraph 4584 Human ACC1 is regulated allosterically, via specific protein–protein interactions, and by reversible phosphorylation. Dynamic polymerization of human ACC1 is linked to increased activity and is regulated allosterically by the activator citrate and the inhibitor palmitate, or by binding of the small protein MIG-12 (ref.). Human ACC1 is further regulated by specific phosphorylation-dependent binding of BRCA1 to Ser1263 in the CD. BRCA1 binds only to the phosphorylated form of ACC1 and prevents ACC activation by phosphatase-mediated dephosphorylation. Furthermore, phosphorylation by AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA) leads to a decrease in ACC1 activity. AMPK phosphorylates ACC1 in vitro at Ser80, Ser1201 and Ser1216 and PKA at Ser78 and Ser1201. However, regulatory effects on ACC1 activity are mainly mediated by phosphorylation of Ser80 and Ser1201 (refs). Phosphorylated Ser80, which is highly conserved only in higher eukaryotes, presumably binds into the Soraphen A-binding pocket. The regulatory Ser1201 shows only moderate conservation across higher eukaryotes, while the phosphorylated Ser1216 is highly conserved across all eukaryotes. However, no effect of Ser1216 phosphorylation on ACC activity has been reported in higher eukaryotes. species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z Human protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:18:39Z regulated allosterically ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:18:58Z regulated allosterically chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T17:19:11Z citrate chemical CHEBI: melaniev@ebi.ac.uk 2023-03-17T17:19:17Z palmitate protein PR: melaniev@ebi.ac.uk 2023-03-17T17:19:24Z MIG-12 species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z Human protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-17T17:01:12Z BRCA1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:19:35Z Ser1263 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD protein PR: melaniev@ebi.ac.uk 2023-03-17T17:01:12Z BRCA1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:24:12Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:24:39Z phosphatase ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-17T17:19:47Z AMP-activated protein kinase protein PR: melaniev@ebi.ac.uk 2023-03-17T17:19:53Z AMPK protein PR: melaniev@ebi.ac.uk 2023-03-17T17:19:56Z cAMP-dependent protein kinase protein PR: melaniev@ebi.ac.uk 2023-03-17T17:20:03Z PKA protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:19:53Z AMPK protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:13Z Ser80 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:20Z Ser1201 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:28Z Ser1216 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:20:03Z PKA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:35Z Ser78 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:20Z Ser1201 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:13Z Ser80 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:20Z Ser1201 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z Phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:13Z Ser80 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:30Z higher eukaryotes site SO: melaniev@ebi.ac.uk 2023-03-17T17:22:35Z Soraphen A-binding pocket residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:20Z Ser1201 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:24:21Z moderate conservation taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:30Z higher eukaryotes protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:28Z Ser1216 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:24:48Z eukaryotes residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:28Z Ser1216 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:24:22Z ACC taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:30Z higher eukaryotes INTRO paragraph 5882 For fungal ACC, neither spontaneous nor inducible polymerization has been detected despite considerable sequence conservation to human ACC1. The BRCA1-interacting phosphoserine position is not conserved in fungal ACC, and no other phospho-dependent protein–protein interactions of fungal ACC have been described. In yeast ACC, phosphorylation sites have been identified at Ser2, Ser735, Ser1148, Ser1157 and Ser1162 (ref.). Of these, only Ser1157 is highly conserved in fungal ACC and aligns to Ser1216 in human ACC1. Its phosphorylation by the AMPK homologue SNF1 results in strongly reduced ACC activity. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 protein PR: melaniev@ebi.ac.uk 2023-06-15T09:02:41Z BRCA1 residue_name SO: melaniev@ebi.ac.uk 2023-03-17T22:44:14Z phosphoserine protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:14Z not conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC site SO: melaniev@ebi.ac.uk 2023-03-17T17:28:30Z phosphorylation sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:39Z Ser2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:45Z Ser735 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:52Z Ser1148 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:29:07Z Ser1162 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:30:29Z aligns to residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:28Z Ser1216 species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human protein PR: melaniev@ebi.ac.uk 2023-03-17T17:18:52Z ACC1 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-22T10:25:15Z AMPK protein PR: melaniev@ebi.ac.uk 2023-03-18T22:55:30Z SNF1 protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:25:26Z ACC INTRO paragraph 6491 Despite the outstanding relevance of ACC in primary metabolism and disease, the dynamic organization and regulation of the giant eukaryotic, and in particular fungal ACC, remain poorly characterized. Here we provide the structure of Saccharomyces cerevisiae (Sce) ACC CD, intermediate- and low-resolution structures of human (Hsa) ACC CD and larger fragments of fungal ACC from Chaetomium thermophilum (Cth; Fig. 1a). Integrating these data with small-angle X-ray scattering (SAXS) and electron microscopy (EM) observations yield a comprehensive representation of the dynamic structure and regulation of fungal ACC. protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:33:50Z structure species MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:05Z Saccharomyces cerevisiae species MESH: melaniev@ebi.ac.uk 2023-03-17T17:33:58Z Sce protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:03Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:34:09Z structures species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human species MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:15Z Hsa protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:19Z larger fragments taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC species MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:25Z Chaetomium thermophilum species MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:32Z Cth experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:39Z small-angle X-ray scattering experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:45Z SAXS experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:52Z electron microscopy experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:58Z EM taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC RESULTS title_1 7107 Results RESULTS title_2 7115 The organization of the yeast ACC CD taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD RESULTS paragraph 7152 First, we focused on structure determination of the 82-kDa CD. The crystal structure of the CD of SceACC (SceCD) was determined at 3.0 Å resolution by experimental phasing and refined to Rwork/Rfree=0.20/0.24 (Table 1). The overall extent of the SceCD is 70 by 75 Å (Fig. 1b and Supplementary Fig. 1a,b), and the attachment points of the N-terminal 26-residue linker to the BCCP domain and the C-terminal CT domain are separated by 46 Å (the N- and C termini are indicated with spheres in Fig. 1b). SceCD comprises four distinct domains, an N-terminal α-helical domain (CDN), and a central four-helix bundle linker domain (CDL), followed by two α–β-fold C-terminal domains (CDC1/CDC2). CDN adopts a letter C shape, where one of the ends is a regular four-helix bundle (Nα3-6), the other end is a helical hairpin (Nα8,9) and the bridging region comprises six helices (Nα1,2,7,10–12). CDL is composed of a small, irregular four-helix bundle (Lα1–4) and tightly interacts with the open face of CDC1 via an interface of 1,300 Å2 involving helices Lα3 and Lα4. CDL does not interact with CDN apart from the covalent linkage and forms only a small contact to CDC2 via a loop between Lα2/α3 and the N-terminal end of Lα1, with an interface area of 400 Å2. CDC1/CDC2 share a common fold; they are composed of six-stranded β-sheets flanked on one side by two long, bent helices inserted between strands β3/β4 and β4/β5. CDC2 is extended at its C terminus by an additional β-strand and an irregular β-hairpin. On the basis of a root mean square deviation of main chain atom positions of 2.2 Å, CDC1/CDC2 are structurally more closely related to each other than to any other protein (Fig. 1c); they may thus have evolved by duplication. Close structural homologues could not be found for the CDN or the CDC domains. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:39:40Z structure determination structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC species MESH: melaniev@ebi.ac.uk 2023-06-15T09:16:24Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:16:33Z CD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:40:02Z experimental phasing experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:40:05Z refined evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:40:23Z Rwork evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:40:34Z Rfree species MESH: melaniev@ebi.ac.uk 2023-06-15T09:15:20Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:15:46Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:40:45Z 26-residue linker structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:41:57Z BCCP structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:19Z CT species MESH: melaniev@ebi.ac.uk 2023-06-15T09:14:36Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:14:51Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:20Z α-helical domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:46Z four-helix bundle linker domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:01Z α–β-fold C-terminal domains structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:43:29Z C shape structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:35Z regular four-helix bundle structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:39Z Nα3-6 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:42Z helical hairpin structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:45Z Nα8,9 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:44:05Z bridging region structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:44:30Z helices structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:44:34Z Nα1,2,7,10–12 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:44:39Z small, irregular four-helix bundle structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:44:42Z Lα1–4 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 site SO: melaniev@ebi.ac.uk 2023-03-17T17:45:12Z interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:16Z helices structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:19Z Lα3 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:22Z Lα4 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:28Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:30Z Lα2/α3 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:40Z Lα1 site SO: melaniev@ebi.ac.uk 2023-03-17T17:45:43Z interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:45:49Z six-stranded β-sheets structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:46:28Z long, bent helices structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:46:32Z strands structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:46:36Z β3/β4 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:46:39Z β4/β5 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:46:42Z extended structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:46:59Z β-strand structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:47:02Z irregular β-hairpin evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:47:04Z root mean square deviation structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:23Z CDC RESULTS title_2 9025 A regulatory loop mediates interdomain interactions structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop RESULTS paragraph 9077 To define the functional state of insect-cell-expressed ACC variants, we employed mass spectrometry (MS) for phosphorylation site detection. In insect-cell-expressed full-length SceACC, the highly conserved Ser1157 is the only fully occupied phosphorylation site with functional relevance in S. cerevisiae. Additional phosphorylation was detected for Ser2101 and Tyr2179; however, these sites are neither conserved across fungal ACC nor natively phosphorylated in yeast. MS analysis of dissolved crystals confirmed the phosphorylated state of Ser1157 also in SceCD crystals. The SceCD structure thus authentically represents the state of SceACC, where the enzyme is inhibited by SNF1-dependent phosphorylation. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:42Z insect-cell-expressed protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:45Z mass spectrometry experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:50Z MS experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:56Z phosphorylation site detection experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:07:01Z insect-cell-expressed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:07:07Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:07:40Z fully occupied site SO: melaniev@ebi.ac.uk 2023-03-17T19:07:22Z phosphorylation site species MESH: melaniev@ebi.ac.uk 2023-03-17T19:07:49Z S. cerevisiae ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:08:02Z Ser2101 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:08:08Z Tyr2179 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:42:02Z neither conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:51:50Z nor natively phosphorylated taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:50Z MS experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:09:56Z dissolved crystals protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 species MESH: melaniev@ebi.ac.uk 2023-06-15T09:17:12Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:17:25Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:25Z crystals species MESH: melaniev@ebi.ac.uk 2023-06-15T09:17:46Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:17:58Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:27Z structure protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC protein PR: melaniev@ebi.ac.uk 2023-03-18T22:55:36Z enzyme protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:10:50Z inhibited ptm MESH: melaniev@ebi.ac.uk 2023-03-17T19:10:35Z SNF1-dependent phosphorylation RESULTS paragraph 9788 In the SceCD crystal structure, the phosphorylated Ser1157 resides in a regulatory 36-amino-acid loop between strands β2 and β3 of CDC1 (Fig. 1b,d), which contains two additional less-conserved phosphorylation sites (Ser1148 and Ser1162) confirmed in yeast, but not occupied here. This regulatory loop wedges between the CDC1 and CDC2 domains and provides the largest contribution to the interdomain interface. The N-terminal region of the regulatory loop also directly contacts the C-terminal region of CDC2 leading into CT. Phosphoserine 1157 is tightly bound by two highly conserved arginines (Arg1173 and Arg1260) of CDC1 (Fig. 1d). Already the binding of phosphorylated Ser1157 apparently stabilizes the regulatory loop conformation; the accessory phosphorylation sites Ser1148 and Ser1162 in the same loop may further modulate the strength of interaction between the regulatory loop and the CDC1 and CDC2 domains. Phosphorylation of the regulatory loop thus determines interdomain interactions of CDC1 and CDC2, suggesting that it may exert its regulatory function by modifying the overall structure and dynamics of the CD. species MESH: melaniev@ebi.ac.uk 2023-06-15T09:19:21Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:19:33Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T19:13:59Z regulatory 36-amino-acid loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T19:14:02Z strands structure_element SO: melaniev@ebi.ac.uk 2023-03-17T19:14:05Z β2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T19:14:07Z β3 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:14:13Z less-conserved site SO: melaniev@ebi.ac.uk 2023-03-17T17:28:30Z phosphorylation sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:52Z Ser1148 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:29:07Z Ser1162 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 site SO: melaniev@ebi.ac.uk 2023-03-17T19:14:31Z interdomain interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:29Z CT residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:56:31Z Phosphoserine 1157 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved residue_name SO: melaniev@ebi.ac.uk 2023-03-17T19:15:15Z arginines residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:15:21Z Arg1173 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:15:27Z Arg1260 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop site SO: melaniev@ebi.ac.uk 2023-03-17T17:28:30Z phosphorylation sites residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:52Z Ser1148 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:29:07Z Ser1162 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T19:16:03Z same loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z Phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:48Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:17Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD RESULTS paragraph 10924 The functional role of Ser1157 was confirmed by an activity assay based on the incorporation of radioactive carbonate into acid non-volatile material. Phosphorylated SceACC shows only residual activity (kcat=0.4±0.2 s−1, s.d. based on five replicate measurements), which increases 16-fold (kcat=6.5±0.3 s−1) after dephosphorylation with λ protein phosphatase. The values obtained for dephosphorylated SceACC are comparable to earlier measurements of non-phosphorylated yeast ACC expressed in E. coli. residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:17:19Z activity assay protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z Phosphorylated protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:17:37Z kcat evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:17:39Z kcat protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T19:18:38Z λ protein phosphatase protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:44Z dephosphorylated protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:51Z non-phosphorylated taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:18:55Z expressed in species MESH: melaniev@ebi.ac.uk 2023-03-17T17:04:43Z E. coli RESULTS title_2 11436 The variable CD is conserved between yeast and human structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:30:43Z conserved taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human RESULTS paragraph 11489 To compare the organization of fungal and human ACC CD, we determined the structure of a human ACC1 fragment that comprises the BT and CD domains (HsaBT-CD), but lacks the mobile BCCP in between (Fig. 1a). An experimentally phased map was obtained at 3.7 Å resolution for a cadmium-derivatized crystal and was interpreted by a poly-alanine model (Fig. 1e and Table 1). Each of the four CD domains in HsaBT-CD individually resembles the corresponding SceCD domain; however, human and yeast CDs exhibit distinct overall structures. In agreement with their tight interaction in SceCD, the relative spatial arrangement of CDL and CDC1 is preserved in HsaBT-CD, but the human CDL/CDC1 didomain is tilted by 30° based on a superposition of human and yeast CDC2 (Supplementary Fig. 1c). As a result, the N terminus of CDL at helix Lα1, which connects to CDN, is shifted by 12 Å. Remarkably, CDN of HsaBT-CD adopts a completely different orientation compared with SceCD. With CDL/CDC1 superposed, CDN in HsaBT-CD is rotated by 160° around a hinge at the connection of CDN/CDL (Supplementary Fig. 1d). This rotation displaces the N terminus of CDN in HsaBT-CD by 51 Å compared with SceCD, resulting in a separation of the attachment points of the N-terminal linker to the BCCP domain and the C-terminal CT domain by 67 Å (the attachment points are indicated with spheres in Fig. 1e). The BT domain of HsaBT-CD consists of a helix that is surrounded at its N terminus by an antiparallel eight-stranded β-barrel. It resembles the BT of propionyl-CoA carboxylase; only the four C-terminal strands of the β-barrel are slightly tilted. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:09Z determined the structure species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:12Z ACC1 fragment structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:37Z BT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:20Z HsaBT-CD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:36:43Z lacks structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:37:59Z BCCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:36:48Z experimentally phased map chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:26:06Z cadmium structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:20Z HsaBT-CD species MESH: melaniev@ebi.ac.uk 2023-06-15T09:12:38Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:12:47Z CD species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:34Z CDs evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:37:08Z structures species MESH: melaniev@ebi.ac.uk 2023-06-15T09:23:04Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:23:14Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:20Z HsaBT-CD species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:37:11Z superposition species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:41Z helix structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:45Z Lα1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD species MESH: melaniev@ebi.ac.uk 2023-06-15T09:13:08Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:13:21Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:37:14Z superposed structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:49Z hinge structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:13:31Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:13:40Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD species MESH: melaniev@ebi.ac.uk 2023-06-15T09:11:43Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:11:56Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:52Z linker structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:41:11Z BCCP domain structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:38:21Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:37Z BT mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:52:57Z helix structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:37:44Z antiparallel eight-stranded β-barrel structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:37Z BT protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:52:14Z propionyl-CoA carboxylase structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:37:52Z strands of the β-barrel RESULTS paragraph 13128 On the basis of MS analysis of insect-cell-expressed human full-length ACC, Ser80 shows the highest degree of phosphorylation (90%). Ser29 and Ser1263, implicated in insulin-dependent phosphorylation and BRCA1 binding, respectively, are phosphorylated at intermediate levels (40%). The highly conserved Ser1216 (corresponding to S. cerevisiae Ser1157), as well as Ser1201, both in the regulatory loop discussed above, are not phosphorylated. However, residual phosphorylation levels were detected for Ser1204 (7%) and Ser1218 (7%) in the same loop. MS analysis of the HsaBT-CD crystallization sample reveals partial proteolytic digestion of the regulatory loop. Accordingly, most of this loop is not represented in the HsaBT-CD crystal structure. The absence of the regulatory loop might be linked to the less-restrained interface of CDL/CDC1 and CDC2 and altered relative orientations of these domains. Besides the regulatory loop, also the phosphopeptide target region for BRCA1 interaction is not resolved presumably because of pronounced flexibility. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:50Z MS experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:41:25Z insect-cell-expressed species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:06Z human protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:07:07Z full-length protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:13Z Ser80 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:56:37Z Ser29 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:19:35Z Ser1263 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T22:44:38Z insulin-dependent phosphorylation protein PR: melaniev@ebi.ac.uk 2023-03-22T10:26:23Z BRCA1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:28Z Ser1216 species MESH: melaniev@ebi.ac.uk 2023-03-17T19:07:49Z S. cerevisiae residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:58Z Ser1157 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:20Z Ser1201 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:44:58Z not phosphorylated ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:45:13Z Ser1204 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:45:23Z Ser1218 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:44:52Z same loop experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:50Z MS mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:32Z crystallization sample structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:45:34Z this loop mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:56:48Z absence of structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:45:49Z less-restrained site SO: melaniev@ebi.ac.uk 2023-03-17T22:45:54Z interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:10:23Z domains structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop site SO: melaniev@ebi.ac.uk 2023-03-17T22:46:18Z phosphopeptide target region protein PR: melaniev@ebi.ac.uk 2023-06-15T09:03:56Z BRCA1 RESULTS paragraph 14183 At the level of isolated yeast and human CD, the structural analysis indicates the presence of at least two hinges, one with large-scale flexibility at the CDN/CDL connection, and one with tunable plasticity between CDL/CDC1 and CDC2, plausibly affected by phosphorylation in the regulatory loop region. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:47:47Z isolated taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:07Z human structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:47:50Z structural analysis structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:11Z hinges structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:01:03Z CDN/CDL connection structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop RESULTS title_2 14487 The integration of CD into the fungal ACC multienzyme structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T22:49:03Z ACC multienzyme RESULTS paragraph 14541 To further obtain insights into the functional architecture of fungal ACC, we characterized larger multidomain fragments up to the intact enzymes. Using molecular replacement based on fungal ACC CD and CT models, we obtained structures of a variant comprising CthCT and CDC1/CDC2 in two crystal forms at resolutions of 3.6 and 4.5 Å (CthCD-CTCter1/2), respectively, as well as of a CthCT linked to the entire CD at 7.2 Å resolution (CthCD-CT; Figs 1a and 2, Table 1). No crystals diffracting to sufficient resolution were obtained for larger BC-containing fragments, or for full-length Cth or SceACC. To improve crystallizability, we generated ΔBCCP variants of full-length ACC, which, based on SAXS analysis, preserve properties of intact ACC (Supplementary Table 1 and Supplementary Fig. 2a–c). For CthΔBCCP, crystals diffracting to 8.4 Å resolution were obtained. However, molecular replacement did not reveal a unique positioning of the BC domain. Owing to the limited resolution the discussion of structures of CthCD-CT and CthΔBCCP is restricted to the analysis of domain localization. Still, these structures contribute considerably to the visualization of an intrinsically dynamic fungal ACC. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:52:24Z larger multidomain fragments protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:17Z intact protein PR: melaniev@ebi.ac.uk 2023-03-17T22:52:31Z enzymes experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:52:34Z molecular replacement taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:45Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:52:38Z CT evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:52:42Z structures mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:52:45Z variant species MESH: melaniev@ebi.ac.uk 2023-03-17T22:52:51Z Cth structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:52:55Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:52:59Z two crystal forms mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:32Z CthCD-CTCter1/2 species MESH: melaniev@ebi.ac.uk 2023-03-17T22:53:06Z Cth structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:53:09Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:24Z CthCD-CT mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:53:33Z larger BC-containing fragments protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:07:07Z full-length species MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:32Z Cth protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:53:49Z improve crystallizability experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:53:52Z generated mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:53:54Z ΔBCCP variants protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:07:07Z full-length protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:00Z SAXS analysis protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:17Z intact protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:09Z CthΔBCCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:54:14Z crystals experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:17Z molecular replacement structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:18Z BC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:54:39Z structures mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:24Z CthCD-CT mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:09Z CthΔBCCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:54:46Z these structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:28Z dynamic taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC RESULTS paragraph 15756 In all these crystal structures, the CT domains build a canonical head-to-tail dimer, with active sites formed by contributions from both protomers (Fig. 2 and Supplementary Fig. 3a). The connection of CD and CT is provided by a 10-residue peptide stretch, which links the N terminus of CT to the irregular β-hairpin/β-strand extension of CDC2 (Supplementary Fig. 3b). The connecting region is remarkably similar in isolated CD and CthCD-CTCter structures, indicating inherent conformational stability. CD/CT contacts are only formed in direct vicinity of the covalent linkage and involve the β-hairpin extension of CDC2 as well as the loop between strands β2/β3 of the CT N-lobe, which contains a conserved RxxGxN motif. The neighbouring loop on the CT side (between CT β1/β2) is displaced by 2.5 Å compared to isolated CT structures (Supplementary Fig. 3c). On the basis of an interface area of ∼600 Å2 and its edge-to-edge connection characteristics, the interface between CT and CD might be classified as conformationally variable. Indeed, the comparison of the positioning of eight instances of the C-terminal part of CD relative to CT in crystal structures determined here, reveals flexible interdomain linking (Fig. 3a). The CDC2/CT interface acts as a true hinge with observed rotation up to 16°, which results in a translocation of the distal end of CDC2 by 8 Å. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:57:28Z crystal structures structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:57:31Z CT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:57:34Z head-to-tail oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:57:36Z dimer site SO: melaniev@ebi.ac.uk 2023-03-17T17:14:31Z active sites oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:57:46Z protomers structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:57:50Z connection structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:57:53Z CT residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:57:57Z 10-residue peptide stretch structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:01Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:05Z irregular β-hairpin/β-strand extension structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:09Z connecting region protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:58:13Z isolated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:58:20Z CthCD-CTCter evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:58:27Z structures structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:04:33Z CD structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:04:42Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:41Z β-hairpin extension structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:45Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:48Z strands β2/β3 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:58:55Z CT N-lobe protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:30:43Z conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:59:03Z RxxGxN motif structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:26Z loop structure_element SO: melaniev@ebi.ac.uk 2023-03-22T10:26:44Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:59:54Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:31Z β1 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:34Z β2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:59:45Z isolated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:59:51Z CT evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:59:48Z structures site SO: melaniev@ebi.ac.uk 2023-03-17T23:00:01Z interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T22:59:58Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:00:14Z CT evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:00:17Z crystal structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T23:00:19Z determined site SO: melaniev@ebi.ac.uk 2023-03-17T23:00:22Z CDC2/CT interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:00:24Z true hinge structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 RESULTS paragraph 17149 The interface between CDC2 and CDL/CDC1, which is mediated by the phosphorylated regulatory loop in the SceCD structure, is less variable than the CD–CT junction, and permits only limited rotation and tilting (Fig. 3b). Analysis of the impact of phosphorylation on the interface between CDC2 and CDL/CDC1 in CthACC variant structures is precluded by the limited crystallographic resolution. However, MS analysis of CthCD-CT and CthΔBCCP constructs revealed between 60 and 70% phosphorylation of Ser1170 (corresponding to SceACC Ser1157). site SO: melaniev@ebi.ac.uk 2023-03-17T23:01:42Z interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:10Z CDC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop species MESH: melaniev@ebi.ac.uk 2023-06-15T09:20:32Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:20:45Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:01:49Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:01:52Z CD–CT junction ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation site SO: melaniev@ebi.ac.uk 2023-03-17T23:01:56Z interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 mutant MESH: melaniev@ebi.ac.uk 2023-03-17T23:02:04Z CthACC variant evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:02:07Z structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:51Z MS mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:24Z CthCD-CT mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:09Z CthΔBCCP ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:02:17Z Ser1170 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 RESULTS paragraph 17691 The CDN domain positioning relative to CDL/CDC1 is highly variable with three main orientations observed in the structures of SceCD and the larger CthACC fragments: CDN tilts, resulting in a displacement of its N terminus by 23 Å (Fig. 4a, observed in both protomers of CthCD-CT and one protomer of CthΔBCCP, denoted as CthCD-CT1/2 and CthΔBCCP1, respectively). In addition, CDN can rotate around hinges in the connection between CDN/CDL by 70° (Fig. 4b, observed in the second protomer of CthΔBCCP, denoted as CthΔBCCP2) and 160° (Fig. 4c, observed in SceCD) leading to displacement of the anchor site for the BCCP linker by up to 33 and 40 Å, respectively. structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:03Z structures species MESH: melaniev@ebi.ac.uk 2023-06-15T09:21:27Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:21:41Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-17T23:04:06Z larger CthACC fragments structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:19Z protomers mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:24Z CthCD-CT oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:36Z protomer mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:09Z CthΔBCCP mutant MESH: melaniev@ebi.ac.uk 2023-03-17T23:04:40Z CthCD-CT1/2 mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:55:14Z CthΔBCCP1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:04:44Z hinges structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:36Z protomer mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:09Z CthΔBCCP mutant MESH: melaniev@ebi.ac.uk 2023-03-17T23:05:20Z CthΔBCCP2 species MESH: melaniev@ebi.ac.uk 2023-06-15T09:22:20Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:22:30Z CD site SO: melaniev@ebi.ac.uk 2023-03-17T23:05:31Z anchor site structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:40Z BCCP linker RESULTS paragraph 18366 Conformational variability in the CD thus contributes considerably to variations in the spacing between the BC and CT domains, and may extend to distance variations beyond the mobility range of the flexibly tethered BCCP. On the basis of the occurrence of related conformational changes between fungal and human ACC fragments, the observed set of conformations may well represent general states present in all eukaryotic ACCs. structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:44Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:06:29Z CT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:14:37Z flexibly tethered structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:06:34Z BCCP taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:07Z human mutant MESH: melaniev@ebi.ac.uk 2023-03-17T23:06:37Z ACC fragments taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs RESULTS title_2 18793 Large-scale conformational variability of fungal ACC taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC RESULTS paragraph 18846 To obtain a comprehensive view of fungal ACC dynamics in solution, we employed SAXS and EM. SAXS analysis of CthACC agrees with a dimeric state and an elongated shape with a maximum extent of 350 Å (Supplementary Table 1). The smooth appearance of scattering curves and derived distance distributions might indicate substantial interdomain flexibility (Supplementary Fig. 2a–c). Direct observation of individual full-length CthACC particles, according to MS results predominantly in a phosphorylated low-activity state, in negative stain EM reveals a large set of conformations from rod-like extended to U-shaped particles. Class averages, obtained by maximum-likelihood-based two-dimensional (2D) classification, are focused on the dimeric CT domain and the full BC–BCCP–CD domain of only one protomer, due to the non-coordinated motions of the lateral BC/CD regions relative to the CT dimer. They identify the connections between CDN/CDL and between CDC2/CT as major contributors to conformational heterogeneity (Supplementary Fig. 4a,b). The flexibility in the CDC2/CT hinge appears substantially larger than the variations observed in the set of crystal structures. The BC domain is not completely disordered, but laterally attached to BT/CDN in a generally conserved position, albeit with increased flexibility. Surprisingly, in both the linear and U-shaped conformations, the approximate distances between the BC and CT active sites would remain larger than 110 Å. These observed distances are considerably larger than in static structures of any other related biotin-dependent carboxylase. Furthermore, based on an average length of the BCCP–CD linker in fungal ACC of 26 amino acids, mobility of the BCCP alone would not be sufficient to bridge the active sites of BC and CT. Consequently, increased flexibility or additional modes of conformational changes may be required for productive catalysis. The most relevant candidate site for mediating such additional flexibility and permitting an extended set of conformations is the CDC1/CDC2 interface, which is rigidified by the Ser1157-phosphorylated regulatory loop, as depicted in the SceCD crystal structure. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T23:11:14Z ACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:20Z in solution experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:45Z SAXS experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:59Z EM experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:45Z SAXS protein PR: melaniev@ebi.ac.uk 2023-03-18T22:55:55Z CthACC oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:25Z dimeric protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:28Z elongated shape evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:30Z scattering curves evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:33Z derived distance distributions protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:07:07Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-18T22:55:55Z CthACC evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:37Z particles experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T19:06:51Z MS protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:39Z low-activity state experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T23:11:42Z negative stain EM protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:45Z rod-like extended protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:48Z U-shaped evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:50Z particles evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:53Z Class averages experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T23:11:55Z maximum-likelihood-based two-dimensional (2D) classification oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:11:58Z dimeric structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:12:01Z CT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:12:03Z full mutant MESH: melaniev@ebi.ac.uk 2023-03-17T23:12:06Z BC–BCCP–CD oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:36Z protomer structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:12:12Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:12:20Z CT oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:12:24Z dimer structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:12:36Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:12:39Z CDC2/CT hinge evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:12:42Z crystal structures structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:12:45Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:38Z BT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:13:12Z generally conserved position protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:13:14Z linear and U-shaped conformations structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:13:17Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:13:20Z CT site SO: melaniev@ebi.ac.uk 2023-03-17T17:14:31Z active sites protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:51:56Z static evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:38Z structures protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T23:13:25Z biotin-dependent carboxylase structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:13:28Z BCCP–CD linker taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:13:31Z 26 amino acids structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:13:34Z BCCP site SO: melaniev@ebi.ac.uk 2023-03-17T17:14:31Z active sites structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:13:39Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T23:13:42Z CT site SO: melaniev@ebi.ac.uk 2023-03-17T23:13:53Z CDC1/CDC2 interface residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop species MESH: melaniev@ebi.ac.uk 2023-06-15T09:24:08Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:24:18Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure DISCUSS title_1 21030 Discussion DISCUSS paragraph 21041 Altogether, the architecture of fungal ACC is based on the central dimeric CT domain (Fig. 4d). The CD consists of four distinct subdomains and acts as a tether from the CT to the mobile BCCP and an oriented BC domain. The CD has no direct role in substrate recognition or catalysis but contributes to the regulation of all eukaryotic ACCs. In higher eukaryotic ACCs, regulation via phosphorylation is achieved by combining the effects of phosphorylation at Ser80, Ser1201 and Ser1263. In fungal ACC, however, Ser1157 in the regulatory loop of the CD is the only phosphorylation site that has been demonstrated to be both phosphorylated in vivo and involved in the regulation of ACC activity. In its phosphorylated state, the regulatory loop containing Ser1157 wedges between CDC1/CDC2 and presumably limits the conformational freedom at this interdomain interface. However, flexibility at this hinge may be required for full ACC activity, as the distances between the BCCP anchor points and the active sites of BC and CT observed here are such large that mobility of the BCCP alone is not sufficient for substrate transfer. The current data thus suggest that regulation of fungal ACC is mediated by controlling the dynamics of the unique CD, rather than directly affecting catalytic turnover at the active sites of BC and CT. A comparison between fungal and human ACC will help to further discriminate mechanistic differences that contribute to the extended control and polymerization of human ACC. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:19:20Z dimeric structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:19:23Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:04Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:53:51Z subdomains structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:19:38Z CT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:19:33Z mobile structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:19:30Z BCCP protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:19:36Z oriented structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:19:27Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:05Z CD taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:52:04Z higher eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:13Z Ser80 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:20:20Z Ser1201 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:19:35Z Ser1263 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:11Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:05Z CD site SO: melaniev@ebi.ac.uk 2023-03-18T22:19:47Z phosphorylation site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated protein_type MESH: melaniev@ebi.ac.uk 2023-03-22T10:27:20Z ACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:20:02Z conformational freedom site SO: melaniev@ebi.ac.uk 2023-03-18T22:20:06Z interdomain interface structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:20:10Z hinge protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:20:42Z full ACC activity structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:20:45Z BCCP anchor points site SO: melaniev@ebi.ac.uk 2023-03-17T17:14:31Z active sites structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:20:51Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:20:53Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:20:56Z BCCP taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:21:01Z unique structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:05Z CD site SO: melaniev@ebi.ac.uk 2023-03-17T17:14:31Z active sites structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:21:06Z BC structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:21:08Z CT taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:07Z human protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC species MESH: melaniev@ebi.ac.uk 2023-03-17T17:06:07Z human protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC DISCUSS paragraph 22541 Most recently, a crystal structure of near full-length non-phosphorylated ACC from S. cerevisae (lacking only 21 N-terminal amino acids, here denoted as flACC) was published by Wei and Tong. In flACC, the ACC dimer obeys twofold symmetry and assembles in a triangular architecture with dimeric BC domains (Supplementary Fig. 5a). In their study, mutational data indicate a requirement for BC dimerization for catalytic activity. The transition from the elongated open shape, observed in our experiments, towards a compact triangular shape is based on an intricate interplay of several hinge-bending motions in the CD (Fig. 4d). Comparison of flACC with our CthΔBCCP structure reveals the CDC2/CT hinge as a major contributor to conformational flexibility (Supplementary Fig. 5b,c). In flACC, CDC2 rotates ∼120° with respect to the CT domain. A second hinge can be identified between CDC1/CDC2. On the basis of a superposition of CDC2, CDC1 of the phosphorylated SceCD is rotated by 30° relative to CDC1 of the non-phosphorylated flACC (Supplementary Fig. 5d), similar to what we have observed for the non-phosphorylated HsaBT-CD (Supplementary Fig. 1d). When inspecting all individual protomer and fragment structures in their study, Wei and Tong also identify the CDN/CDC1 connection as a highly flexible hinge, in agreement with our observations. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:24:18Z near full-length protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:51Z non-phosphorylated protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC species MESH: melaniev@ebi.ac.uk 2023-03-18T22:56:21Z S. cerevisae protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:24:24Z lacking only residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:56:58Z 21 mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:24:50Z dimer protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:24:53Z triangular architecture oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:24:55Z dimeric structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:24:58Z BC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:25:00Z mutational data protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:25:03Z elongated open shape protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:25:06Z compact triangular shape structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:05Z CD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:25:09Z Comparison mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:09Z CthΔBCCP evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:25:18Z structure structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:25:21Z CDC2/CT hinge mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:25:27Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:25:30Z second hinge structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:25:32Z superposition structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated species MESH: melaniev@ebi.ac.uk 2023-06-15T09:25:04Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:25:23Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:51Z non-phosphorylated mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:51Z non-phosphorylated mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:26:16Z inspecting oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:26:34Z protomer mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:26:57Z fragment evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:27:08Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:27:12Z CDN/CDC1 connection protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:27:14Z highly flexible structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:27:17Z hinge DISCUSS paragraph 23895 The only bona fide regulatory phophorylation site of fungal ACC in the regulatory loop is directly participating in CDC1/CDC2 domain interactions and thus stabilizes the hinge conformation. In flACC, the regulatory loop is mostly disordered, illustrating the increased flexibility due to the absence of the phosphoryl group. Only in three out of eight observed protomers a short peptide stretch (including Ser1157) was modelled. In those instances the Ser1157 residue is located at a distance of 14–20 Å away from the location of the phosphorylated serine observed here, based on superposition of either CDC1 or CDC2. Applying the conformation of the CDC1/CDC2 hinge observed in SceCD on flACC leads to CDN sterically clashing with CDC2 and BT/CDN clashing with CT (Supplementary Fig. 6a,b). Thus, in accordance with the results presented here, phosphorylation of Ser1157 in SceACC most likely limits flexibility in the CDC1/CDC2 hinge such that activation through BC dimerization is not possible (Fig. 4d), which however does not exclude intermolecular dimerization. In addition, EM micrographs of phosphorylated and dephosphorylated SceACC display for both samples mainly elongated and U-shaped conformations and reveal no apparent differences in particle shape distributions (Supplementary Fig. 7). This implicates that the triangular shape with dimeric BC domains has a low population also in the active form, even though a biasing influence of grid preparation cannot be excluded completely. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:29:53Z regulatory site SO: melaniev@ebi.ac.uk 2023-03-18T22:29:56Z phophorylation site taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:05:34Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:05:42Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:30:03Z hinge conformation mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:30:08Z mostly disordered chemical CHEBI: melaniev@ebi.ac.uk 2023-03-18T22:30:13Z phosphoryl oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:30:17Z protomers structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:30:20Z short peptide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:30:23Z modelled residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name SO: melaniev@ebi.ac.uk 2023-03-17T17:08:22Z serine experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:30:36Z superposition structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:30:39Z Applying structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:30:51Z CDC1/CDC2 hinge species MESH: melaniev@ebi.ac.uk 2023-06-15T09:25:49Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:25:58Z CD mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:24:36Z flACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:07:38Z BT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:26Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:30:58Z CT ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:31:03Z CDC1/CDC2 hinge structure_element SO: melaniev@ebi.ac.uk 2023-03-22T10:27:50Z BC experimental_method MESH: melaniev@ebi.ac.uk 2023-03-17T17:34:59Z EM evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:31:06Z micrographs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:44Z dephosphorylated protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:31:10Z elongated and U-shaped conformations evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:31:12Z particle shape distributions protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:31:14Z triangular shape oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:31:17Z dimeric structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:31:20Z BC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:31:23Z active form DISCUSS paragraph 25397 Large-scale conformational variability has also been observed in most other carrier protein-based multienzymes, including polyketide and fatty-acid synthases (with the exception of fungal-type fatty-acid synthases), non-ribosomal peptide synthetases and the pyruvate dehydrogenase complexes, although based on completely different architectures. Together, this structural information suggests that variable carrier protein tethering is not sufficient for efficient substrate transfer and catalysis in any of these systems. The determination of a set of crystal structures of SceACC in two states, unphosphorylated and phosphorylated at the major regulatory site Ser1157, provides a unique depiction of multienzyme regulation by post-translational modification (Fig. 4d). The phosphorylated regulatory loop binds to an allosteric site at the interface of two non-catalytic domains and restricts conformational freedom at several hinges in the dynamic ACC. It disfavours the adoption of a rare, compact conformation, in which intramolecular dimerization of the BC domains results in catalytic turnover. The regulation of activity thus results from restrained large-scale conformational dynamics rather than a direct or indirect influence on active site structure. To our best knowledge, ACC is the first multienzyme for which such a phosphorylation-dependent mechanical control mechanism has been visualized. However, the example of ACC now demonstrates the possibility of regulating activity by controlled dynamics of non-enzymatic linker regions also in other families of carrier-dependent multienzymes. Understanding such structural and dynamic constraints imposed by scaffolding and linking in carrier protein-based multienzyme systems is a critical prerequisite for engineering of efficient biosynthetic assembly lines. protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:33:37Z carrier protein-based multienzymes protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:33:40Z polyketide and fatty-acid synthases protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:33:43Z fungal-type fatty-acid synthases protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:33:46Z non-ribosomal peptide synthetases protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:33:48Z pyruvate dehydrogenase complexes evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:33:52Z structural information experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:33:54Z determination of a set of evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:42Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-17T17:39:48Z SceACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:33:58Z unphosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated site SO: melaniev@ebi.ac.uk 2023-03-18T22:34:01Z major regulatory site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop site SO: melaniev@ebi.ac.uk 2023-03-18T22:34:07Z allosteric site site SO: melaniev@ebi.ac.uk 2023-03-18T22:34:25Z interface protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:34:56Z non-catalytic structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:35:18Z hinges protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:55:28Z dynamic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:35:47Z rare, compact conformation structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:35:50Z BC site SO: melaniev@ebi.ac.uk 2023-03-18T22:35:54Z active site structure protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:36:05Z multienzyme ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:52Z phosphorylation protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:46Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:36:10Z non-enzymatic linker regions protein_type MESH: melaniev@ebi.ac.uk 2023-03-18T22:36:08Z carrier-dependent multienzymes METHODS title_1 27220 Methods METHODS title_2 27228 Protein expression and purification METHODS paragraph 27264 All proteins were expressed in the Baculovirus Expression Vector System. The MultiBac insect cell expression plasmid pACEBACI (Geneva Biotech) was modified to host a GATEWAY (LifeTechnologies) cassette with an N-terminal 10xHis-tag, named pAB1GW-NH10 hereafter. Full-length HsaACC (Genebank accession #Q13085), SceACC (#Q00955) and CthACC (#G0S3L5) were cloned into pAB1GW-NH10 using GATEWAY according to the manufacturer's manual. Truncated variants were constructed by PCR amplification, digestion of the template DNA with DpnI, phosphorylation of the PCR product and religation of the linear fragment to a circular plasmid. The following constructs were used for this study: SceACC (1–2,233), CthACC (1–2,297), CthΔBCCP (1–2,297, Δ700–765), CthCD-CT (788–2,297), CthCD-CTCter (1,114–2,297), SceCD (768–1,494) and HsaBT-CD (622–1,584, Δ753–818). Bacmid and virus production was carried out according to MultiBac instructions. Baculovirus generation and amplification as well as protein expression were performed in Sf21 cells (Expression Systems) in Insect-Xpress medium (Lonza). The cells were harvested 68–96 h post infection by centrifugation and stored at −80 °C until being processed. METHODS paragraph 28486 Cells were lysed by sonication and the lysate was cleared by ultracentrifugation. Soluble protein was purified using Ni-NTA (Genscript) and size exclusion chromatography (Superose 6, GE Healthcare). The affinity tag was removed by tobacco etch virus (TEV) protease cleavage overnight at 4 °C. TEV protease and uncleaved protein were removed by orthogonal Ni-NTA purification before size exclusion chromatography. SceACC, CthACC and CthΔBCCP were further purified by high-resolution anion exchange chromatography before size exclusion chromatography. Purified SceCD, CthCD-CTCter, CthCD-CT, CthΔBCCP, CthACC and SceACC were concentrated to 10 mg ml−1 in 30 mM 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Purified HsaBT-CD was concentrated to 20 mg ml−1 in 20 mM bicine pH 8.0, 200 mM NaCl, 5% glycerol and 5 mM tris(2-carboxyethyl) phosphine (TCEP). Proteins were used directly or were stored at −80 °C after flash-freezing in liquid nitrogen. METHODS title_2 29533 Protein crystallization METHODS paragraph 29557 All crystallization experiments were conducted using sitting drop vapour diffusion. SceCD crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M BisTrisPropane pH 6.5, 0.05–0.2 M di-sodium malonate, 20–30% polyethylene glycol (PEG) 3350, 10 mM trimethylamine or 2% benzamidine) in a 1:1 or 2:1 ratio. Crystals appeared after several days and continued to grow for 20–200 days. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol and flash-cooled in liquid nitrogen. For heavy metal derivatization the crystals were incubated in stabilization solution supplemented with 1 mM Thimerosal or 10 mM EuCl2, and then backsoaked for 15 s in stabilization solution without heavy metal. METHODS paragraph 30328 Initial crystals of HsaBT-CD grew in 0.1 M Tris pH 8.5, 0.35 M tri-potassium citrate and 2–3.5% PEG10000 at 19 °C. After several rounds of optimization, good-quality diffraction crystals were obtained at 19 °C in 0.1 M MES pH 6, 0.25–0.35 M tri-potassium citrate, 2–5% PEG10000 and 0.01–0.04 M cadmium chloride. The protein drop contained a 1:1 ratio of protein and reservoir solution. Crystals grew immediately and stopped growing after 3 days. They were dehydrated and cryoprotected in several steps in artificial mother liquor containing incrementally increasing concentrations of tri-potassium citrate, PEG10000 and ethylene glycol and then flash-cooled in liquid nitrogen. The final solution was composed of 0.1 M MES pH 6, 0.5 M tri-potassium citrate, 6.75% PEG10000, 0.01 M cadmium chloride and 22% ethylene glycol. METHODS paragraph 31179 CthCD-CTCter crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M HEPES pH 7.5, 2–7% Tacsimate pH 7, 7.5–15% PEG monomethyl ether 5000) in a 1:1 ratio. Crystals appeared after several days and continued to grow for up to 2 weeks. Crystals were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol. METHODS paragraph 31546 CthCD-CT ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Bicine pH 8.5–9.5, 4–8% PEG8000) in a 1:1 or 1:2 ratio. Crystals grew 8– 10 days and were cryoprotected by short incubation in mother liquor supplemented with 22% ethylene glycol before flash-cooling in liquid nitrogen. METHODS paragraph 31867 CthΔBCCP ACC crystals were grown at 19 °C by mixing protein and reservoir solution (0.1 M Morpheus buffer 3 (Molecular Dimensions, MD2-100-102), 7–12% Morpheus ethylene glycols mix (MD2-100-74), 8–12% PEG4000, 17–23% glycerol) in a 1:1 or 1:2 ratio. Crystals grew up to 3 weeks and were cryoprotected in reservoir solution before flash-cooling in liquid nitrogen. METHODS title_2 32243 Structure determination and analysis of phosphorylation METHODS paragraph 32299 All X-ray diffraction data were collected at beamlines X06SA (PXI) or X06DA (PXIII) at the Swiss Light Source (SLS, Paul Scherrer Institute, Villigen, Switzerland) equipped with PILATUS detectors. The wavelength of data collection was 1.000 Å for native crystals, and 1.527 and 1.907 Å for crystals derivatized with europium and cadmium, respectively. Raw data were processed using XDS. Molecular replacement was carried out using Phaser 2.5.7 and 2.6.0, density modification was performed using Parrot and resolve, multicrystal averaging was carried out using phenix. All model building procedures were conducted using Coot and figures were prepared using PyMOL (Schrödinger LLC). METHODS paragraph 32988 Diffraction of initial SceCD crystals in space group P43212 with unit cell dimensions of a=b=110.3 Å and c=131.7 Å was limited to 3.5 Å. The resolution was improved to 3 Å by addition of trimethylamine or benzamidine to the reservoir solution without significant changes in unit cell dimensions. Crystals derivatized with thimerosal and europium were used for initial SAD phase determination using the SHELXC/D package. Two mercury and four europium sites were located, and an initial model was placed in the resulting maps. Since crystals derivatized with europium were slightly non-isomorphous with a c axis length of 127 Å, multicrystal averaging was used for density modification and provided directly interpretable maps. Iterative cycles of model building and refinement in Buster (version 2.10.2; Global Phasing Ltd) converged at Rwork/Rfree of 0.20/0.24. The final model lacks the disordered N terminus (amino acids 768–789), an extended loop in the CDC1 domain (1,203–1,215), a short stretch (1,147–1,149) preceding the regulatory loop and the two very C-terminal residues (1,493–1,494). On the basis of temperature factor analysis, the start and end of the regulatory loop show higher disorder than the region around the interacting phosphoserine 1157. MS analysis of dissolved crystals detected quantitative phosphorylation of the regulatory Ser1157, as also found for full-length SceACC, and additionally albeit with much lower occurrence, phosphorylation of Ser790, Ser1137, Ser1148 and Ser1159. A modelled phosphoryl position for Ser1159 could overlap with the one of Ser1157, and might be represented in the crystal. For all other phosphorylation sites no difference density could be observed, probably because of very low occupancy. PDBeFold was used to search for structural homologues. The thresholds for lowest acceptable percentage of matched secondary structure elements were 70% for the search query and 20% for the result. METHODS paragraph 34956 Initial HsaBT-CD crystals were obtained in space group I4122 with a=b=240.1 Å and c=768.9 Å and diffracted to 7.5 Å. Optimized and dehydrated crystals also belonged to space group I4122 but with unit cell parameters a=b=267.3 Å and c=210.6 Å and diffracted to a resolution of 3.7 Å. Phase information was obtained from SAD based on bound cadmium ions from the crystallization condition. Six cadmium positions were located in a 4.0-Å resolution data set at 1.9 Å wavelength using SHELXC/D via the HKL2MAP interface. Density modification and phasing based on this anomalous data set, a 3.7-Å resolution data set at 1.0 Å wavelength and additional non-isomorphous lower-resolution data sets led to a high-quality electron density map. At the intermediate resolution obtained, the map was interpreted by a poly-alanine model, which was guided by predicted secondary structure as well as sequence and structural alignment with SceCD. The final model contains five cadmium ions and refines using phenix against experimental data with Rwork/Rfree of 0.35/0.38, as expected for a poly-alanine model. Two HsaBT-CD monomers are packed in the asymmetric unit via the CDN and BT domains. Density on top of the β-barrel of one BT most likely representing parts of the BT–CD linker guided the assignment of this BT to its linked CD partner domain. This BT-to-CD assignment was further supported by the analysis of an additional lower-resolution crystal form. Cadmium ions were found to participate in crystal packing. METHODS paragraph 36489 In HsaACC, phosphorylation at regulatory sites was detected as provided in the main text. No phosphorylation was detected for other phosphosites previously identified in large-scale phosphoproteomics studies, namely serines 5, 23, 25, 48, 53, 78, 488, 786, 1273 (refs). METHODS paragraph 36759 Two different crystal forms were obtained for CthCD-CTCter (denoted as CthCD-CTCter1 and CthCD-CTCter2), diffracting to 3.6 and 4.5 Å. Both forms packed in space group P212121 with unit cell constants of a=97.7 Å, b=165.3 Å and c=219.2 Å or a=100.2 Å, b=153.5 Å and c=249.2 Å, respectively. Phases were determined by molecular replacement using a homology model based on SceCT (pdb 1od2) as search model in Phaser; multicrystal averaging was applied in density modification. The CT domain was rebuilt and an initial homology model based on the SceCD structure was fitted into difference density for CthCD-CTCter1. Iterative cycles of rebuilding and refinement in Buster converged at Rwork/Rfree of 0.20/0.24. The refined CD fragment served as a starting model for rebuilding CthCD-CTCter2 at lower resolution. Coordinate refinement in Buster was additionally guided by reference model restraints and converged at Rwork/Rfree of 0.24/0.24. Residues 1,114–1,185, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were disordered in both crystal forms and are not included in the models. Helical regions C terminal to Glu2264 of both protomers of CthCD-CTCter1 and C terminal to Leu2259 and Arg2261 of the two protomers of CthCD-CTCter2, respectively, could not be built unambiguously and were therefore interpreted by placing poly-alanine stretches. Conservation was mapped on the CthCD-CTCter1 crystal structure using al2co based on a sequence alignment of 367 fungal ACC sequences calculated by Clustal Omega. MS analysis of purified protein detected 7% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). METHODS paragraph 38412 CthCD-CT crystallized in space group P31212 with unit cell constants of a=b=195.0 Å and c=189.5 Å and crystals diffracted to a resolution of 7.2 Å. The structure was solved by molecular replacement using a model composed of CthCT and CDC2 as search model in Phaser. CDC1 and CDN were placed manually into the resulting maps, and the model was refined using rigid-body, domain-wise TLS and B-factor refinement and NCS- and reference model-restrained coordinate refinement in Buster to Rwork/Rfree of 0.23/0.25. Owing to the low resolution, the maximum allowed B-factor in Buster refinement was increased from the default value of 300–500 Å2, minimizing B-factor clipping to 5% of all atoms. Residues 1,033–1,035, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the models. Helical regions C terminal to Leu2259 and Arg2261 on the two protomers, respectively, were interpreted as described for CthCD-CTCter. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 60% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). Conservation was mapped on the CthCD-CT crystal structure as for CthCD-CTCter. METHODS paragraph 39659 CthΔBCCP ACC crystallized in space group P6422 with unit cell constants of a=b=462.2 Å and c=204.6 Å, resolution was limited to 8.4 Å. Structure determination and refinement was performed as for CthCD-CT, with a maximum allowed B-factor of 500 Å2, minimizing B-factor clipping to 3% of all atoms. Although substantial difference density is observed, no defined positions of the BT and BC domains could be derived because of disorder or partial in situ proteolysis or combinations thereof. In addition, residues 1,032–1,039, 1,134–1,152, 1,213–1,252, 1,380–1,385, 1,465–1,468 and 2,188–2,195 were not included in the model. The MissingAtom macro implemented in Buster was employed to account for missing atoms, the final Rwork/Rfree were 0.30/0.32. A region C terminal to Leu2259 on one protomer was interpreted as poly-alanine. Loop conformations, including the regulatory loop, were modelled as observed in SceCD. MS analysis of purified protein detected 70% phosphorylation at Ser1170 (corresponding to Ser1157 in SceCD). METHODS title_2 40708 Small-angle X-ray scattering METHODS paragraph 40737 Proteins were thawed on ice and dialysed overnight against 30 mM MOPS pH 7, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Raw scattering data were measured at SAXS beamline B21 at Diamond Light Source. The samples were measured at concentrations of 2.5, 5 and 10 mg ml−1. Data were processed using the ATSAS package according to standard procedures. A slight increase in scattering in the very low-resolution range was observed with increasing protein concentrations, which may be because of interparticle attraction or minor aggregation. Scattering intensities were thus extrapolated to zero concentration using point-wise extrapolation implemented in Primus. Direct comparison of raw scattering curves demonstrates the similarity of CthACC and CthΔBCCP, and the derived values such as Rg and Porod Volume match within expected error margins. Molecular mass estimations based on the SAXS–MOW method derive values of 534.7 and 534.0 kDa for CthACC and CthΔBCCP, respectively. The relative discrepancies to the theoretical weights of 516.8 kDa (CthACC) and 503.0 kDa (CthΔBCCP) are 3.5% and 6.2%, respectively, which is in a typical range for this method. METHODS title_2 41932 Electron microscopy METHODS paragraph 41952 Full-length CthACC was diluted to 0.01 mg ml−1 in 30 mM MOPS pH 7.0, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Protein sample was adsorbed to a 200-μm copper grid and stained with 2% uranyl acetate. Grids of CthACC were imaged on a CM-200 microscope (Philips) equipped with a TVIPS F416 4k CMOS camera (Tietz Video and Image Processing Systems). The voltage used was 200 kV, and a magnification of × 50,000 results in a pixel size of 2.14 Å. Initial image processing and particle picking was carried out using Xmipp. Overall, 22,309 particles were picked semi-automatically from 236 micrographs with a box size of 300 × 300 pixels. After extraction, particles with a z-score of more than three were discarded and 22,257 particles were aligned and classified into 48 2D class averages using the maximum-likelihood target function in Fourier space (MLF2D). After 72 iterations, 4,226 additional particles were discarded and the remaining 18,031 particles were re-aligned and classified into 36 classes using MLF2D with a high-resolution cutoff of 30 Å. After 44 iterations the alignment converged and class averages were extracted. METHODS title_2 43128 In vitro biotinylation and activity assay METHODS paragraph 43170 To ensure full functionality, SceACC was biotinylated in vitro using the E. coli biotin ligase BirA. The reaction mixture contained 10 μM ACC, 3.7 μM BirA, 50 mM Tris-HCl, pH 8, 5.5 mM MgCl2, 0.5 mM biotin, 60 mM NaCl, 3 mM ATP and 10% glycerol, and the reaction was allowed to proceed for 7 h at 30 °C. METHODS paragraph 43495 The catalytic activity of phosphorylated and dephosphorylated SceACC was measured by following the incorporation of radioactive 14C into acid-stable non-volatile material. Dephosphorylated ACC was prepared by overnight treatment with λ protein phosphatase (New England Biolabs) of partially purified ACC before the final gel filtration step. The removal of the phosphoryl group from Ser1157 was confirmed by MS. The reaction mixture contained 0.5 μg recombinant ACC in 100 mM potassium phosphate, pH 8, 3 mM ATP, 5 mM MgCl2, 50 mM NaH14CO3 (specific activity 7.4 MBq mmol−1) and 1 mM acetyl-CoA in a total reaction volume of 100 μl. The reaction mixture was incubated for 15 min at 30 °C, stopped by addition of 200 μl 6 M HCl and subsequently evaporated to dryness at 85 °C. The non-volatile residue was redissolved in 100 μl of water, 1 ml Ultima Gold XR scintillation medium (Perkin Elmer) was added and the 14C radioactivity was measured in a Packard Tricarb 2000CA liquid scintillation analyser. Measurements were carried out in five replicates and catalytic activities were calculated using a standard curve derived from measurements of varying concentrations of NaH14CO3 in reaction buffer. METHODS title_1 44734 Additional information METHODS paragraph 44757 Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5I6E (SceCD), 5I87 (HsaBT-CD), 5I6F/5I6G (CthCD-CTCter1/2), 5I6H (CthCD-CT) and 5I6I (CthΔBCCP). METHODS paragraph 44982 How to cite this article: Hunkeler, M. et al. The dynamic organization of fungal acetyl-CoA carboxylase. Nat. 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Biol. ref 116 1996 50804 Xmipp: an image processing package for electron microscopy SUPPL footnote 50863 Author contributions M.H. cloned, expressed, purified and crystallized fungal ACC constructs, determined their structure and carried out SAXS analysis. E.S. cloned, expressed and crystallized human ACC CD and determined its structure. EM analysis was carried out by E.S., M.H. and A.H. S.I. contributed to structural analysis and figure preparation. T.M. designed and supervised work and analysed crystallographic data; all authors contributed to manuscript preparation. ncomms11196-f1.jpg f1 FIG fig_title_caption 51334 The phosphorylated central domain of yeast ACC. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:43:06Z central domain taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:22Z yeast protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:47Z ACC ncomms11196-f1.jpg f1 FIG fig_caption 51382 (a) Schematic overview of the domain organization of eukaryotic ACCs. Crystallized constructs are indicated. (b) Cartoon representation of the SceCD crystal structure. CDN is linked by a four-helix bundle (CDL) to two α–β-fold domains (CDC1 and CDC2). The regulatory loop is shown as bold cartoon, and the phosphorylated Ser1157 is marked by a red triangle. The N- and C termini are indicated by spheres. (c) Superposition of CDC1 and CDC2 reveals highly conserved folds. (d) The regulatory loop with the phosphorylated Ser1157 is bound into a crevice between CDC1 and CDC2, the conserved residues Arg1173 and Arg1260 coordinate the phosphoryl-group. (e) Structural overview of HsaBT-CD. The attachment points to the N-terminal BCCP domain and the C-terminal CT domain are indicated with spheres. All colourings are according to scheme a. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:05:54Z eukaryotic protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:51:46Z ACCs evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:45:28Z Crystallized constructs species MESH: melaniev@ebi.ac.uk 2023-06-15T09:26:45Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:26:57Z CD evidence DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:14Z crystal structure structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:27Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:54:05Z four-helix bundle structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:53Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:54:09Z two α–β-fold domains structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:45:17Z Superposition structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:22:23Z highly conserved structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:54:13Z folds structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:30:43Z conserved residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:15:21Z Arg1173 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:15:27Z Arg1260 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-18T22:45:05Z phosphoryl mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:36:21Z HsaBT-CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:45:08Z BCCP structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:45:03Z CT ncomms11196-f2.jpg f2 FIG fig_title_caption 52228 Architecture of the CD–CT core of fungal ACC. structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:05Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:45:52Z CT taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:47Z ACC ncomms11196-f2.jpg f2 FIG fig_caption 52276 Cartoon representation of crystal structures of multidomain constructs of CthACC. One protomer is shown in colour and one in grey. Individual domains are labelled; the active site of CT and the position of the conserved regulatory phosphoserine site based on SceCD are indicated by an asterisk and a triangle, respectively. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:54:47Z crystal structures mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:46:20Z multidomain constructs protein PR: melaniev@ebi.ac.uk 2023-03-18T22:55:55Z CthACC oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:36Z protomer site SO: melaniev@ebi.ac.uk 2023-03-18T22:46:26Z active site structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:46:28Z CT protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T22:30:43Z conserved protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:52:00Z regulatory site SO: melaniev@ebi.ac.uk 2023-03-18T22:46:31Z phosphoserine site species MESH: melaniev@ebi.ac.uk 2023-06-15T09:27:38Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:27:49Z CD ncomms11196-f3.jpg f3 FIG fig_title_caption 52600 Variability of the connections of CDC2 to CT and CDC1 in fungal ACC. structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:47:01Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:47Z ACC ncomms11196-f3.jpg f3 FIG fig_caption 52669 (a) Hinge properties of the CDC2–CT connection analysed by a CT-based superposition of eight instances of the CDC2-CT segment. For clarity, only one protomer of CthCD-CTCter1 is shown in full colour as reference. For other instances, CDC2 domains are shown in transparent tube representation with only one helix each highlighted. The range of hinge bending is indicated and the connection points between CDC2 and CT (blue) as well as between CDC1 and CDC2 (green and grey) are marked as spheres. (b) The interdomain interface of CDC1 and CDC2 exhibits only limited plasticity. Representation as in a, but the CDC1 and CDC2 are superposed based on CDC2. One protomer of CthΔBCCP is shown in colour, the CDL domains are omitted for clarity and the position of the phosphorylated serine based on SceCD is indicated with a red triangle. The connection points from CDC1 to CDC2 and to CDL are represented by green spheres. structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:48:32Z Hinge structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:48:30Z CDC2–CT connection experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:48:55Z CT-based superposition mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:48:45Z CDC2-CT segment oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:36Z protomer mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:55:19Z CthCD-CTCter1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:49:08Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:18Z CDC2 site SO: melaniev@ebi.ac.uk 2023-03-18T22:48:49Z interdomain interface structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:19Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:19Z CDC2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:48:52Z superposed structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:19Z CDC2 oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T23:04:36Z protomer mutant MESH: melaniev@ebi.ac.uk 2023-03-17T22:54:10Z CthΔBCCP structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:54Z CDL protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated residue_name SO: melaniev@ebi.ac.uk 2023-03-17T17:08:22Z serine species MESH: melaniev@ebi.ac.uk 2023-06-15T09:28:56Z Sce structure_element SO: melaniev@ebi.ac.uk 2023-06-15T09:29:06Z CD structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:19Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:54Z CDL ncomms11196-f4.jpg f4 FIG fig_title_caption 53591 The conformational dynamics of fungal ACC. taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:47Z ACC ncomms11196-f4.jpg f4 FIG fig_caption 53634 (a–c) Large-scale conformational variability of the CDN domain relative to the CDL/CDC1 domain. CthCD-CT1 (in colour) serves as reference, the compared structures (as indicated, numbers after construct name differentiate between individual protomers) are shown in grey. Domains other than CDN and CDL/CDC1 are omitted for clarity. The domains are labelled and the distances between the N termini of CDN (spheres) in the compared structures are indicated. (d) Schematic model of fungal ACC showing the intrinsic, regulated flexibility of CD in the phosphorylated inhibited or the non-phosphorylated activated state. Flexibility of the CDC2/CT and CDN/CDL hinges is illustrated by arrows. The Ser1157 phosphorylation site and the regulatory loop are schematically indicated in magenta. structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:27Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:54Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 mutant MESH: melaniev@ebi.ac.uk 2023-03-18T22:55:24Z CthCD-CT1 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-18T22:50:49Z compared structures oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:50:52Z protomers structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:27Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:54Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:11Z CDC1 structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:27Z CDN taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:48:12Z fungal protein_type MESH: melaniev@ebi.ac.uk 2023-03-17T16:55:47Z ACC structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:05Z CD protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T16:52:59Z phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:50:57Z inhibited protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-17T19:18:51Z non-phosphorylated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-18T22:51:00Z activated structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:43:19Z CDC2 structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:51:03Z CT structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:27Z CDN structure_element SO: melaniev@ebi.ac.uk 2023-03-17T17:42:54Z CDL structure_element SO: melaniev@ebi.ac.uk 2023-03-18T22:51:06Z hinges residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-17T17:28:59Z Ser1157 ptm MESH: melaniev@ebi.ac.uk 2023-03-17T17:07:53Z phosphorylation structure_element SO: melaniev@ebi.ac.uk 2023-03-17T16:52:49Z regulatory loop t1.xml t1 TABLE table_title_caption 54420 Crystallographic data collection and refinement statistics. t1.xml t1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><italic><bold>Sce</bold></italic><bold>CD</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Sce</bold></italic><bold>CD Thimerosal</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Sce</bold></italic><bold>CD Eu</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Hsa</bold></italic><bold>BT-CD</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Hsa</bold></italic><bold>BT-CD Cd</bold>^{<bold>2+</bold>}</th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic><bold>CD-CT</bold>_{<bold>Cter1</bold>}</th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic><bold>CD-CT</bold>_{<bold>Cter2</bold>}</th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic><bold>CD-CT</bold></th><th align="center" valign="top" charoff="50"><italic><bold>Cth</bold></italic>Δ<bold>BCCP</bold></th></tr></thead><tbody valign="top"><tr><td colspan="10" align="left" valign="top" charoff="50"><italic>Data collection</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50">P4₃2₁2</td><td align="center" valign="top" charoff="50">P4₃2₁2</td><td align="center" valign="top" charoff="50">P4₃2₁2</td><td align="center" valign="top" charoff="50">I4₁22</td><td align="center" valign="top" charoff="50">I4₁22</td><td align="center" valign="top" charoff="50">P2₁2₁2₁</td><td align="center" valign="top" charoff="50">P2₁2₁2₁</td><td align="center" valign="top" charoff="50">P3₁2₁2</td><td align="center" valign="top" charoff="50">P6₄22</td></tr><tr><td align="left" valign="top" charoff="50"> Cell dimensions</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  <italic>a, b, c</italic> (Å)</td><td align="center" valign="top" charoff="50">110.86, 110.86, 131.12</td><td align="center" valign="top" charoff="50">111.22, 111.22, 131.49</td><td align="center" valign="top" charoff="50">108.65, 108.65, 127.36</td><td align="center" valign="top" charoff="50">267.27, 267.27, 210.61</td><td align="center" valign="top" charoff="50">267.67, 267.67, 210.46</td><td align="center" valign="top" charoff="50">97.66, 165.34, 219.23</td><td align="center" valign="top" charoff="50">100.17, 153.45, 249,24</td><td align="center" valign="top" charoff="50">295.02, 295.02, 189.52</td><td align="center" valign="top" charoff="50">462.20, 462.20, 204.64</td></tr><tr><td align="left" valign="top" charoff="50">  α, β, γ (°)</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 120</td><td align="center" valign="top" charoff="50">90, 90, 120</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution<xref ref-type="fn" rid="t1-fn1">*</xref> (Å)</td><td align="center" valign="top" charoff="50">3.0</td><td align="center" valign="top" charoff="50">3.4</td><td align="center" valign="top" charoff="50">4.0</td><td align="center" valign="top" charoff="50">3.7</td><td align="center" valign="top" charoff="50">4.1</td><td align="center" valign="top" charoff="50">3.6</td><td align="center" valign="top" charoff="50">4.5</td><td align="center" valign="top" charoff="50">7.2</td><td align="center" valign="top" charoff="50">8.4</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic>_Merge<xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">18.2 (389.6)</td><td align="center" valign="top" charoff="50">20.5 (306.1)</td><td align="center" valign="top" charoff="50">40.6 (327.0)</td><td align="center" valign="top" charoff="50">7.5 (400.9)</td><td align="center" valign="top" charoff="50">15 (730.5)</td><td align="center" valign="top" charoff="50">14.5 (384.5)</td><td align="center" valign="top" charoff="50">27.4 (225.6)</td><td align="center" valign="top" charoff="50">5.6 (302.6)</td><td align="center" valign="top" charoff="50">29.4 (381.7)</td></tr><tr><td align="left" valign="top" charoff="50"> CC ½<xref ref-type="fn" rid="t1-fn1">*</xref><xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">100 (58.3)</td><td align="center" valign="top" charoff="50">99.9 (42.6)</td><td align="center" valign="top" charoff="50">99.9 (48.5)</td><td align="center" valign="top" charoff="50">100 (59.4)</td><td align="center" valign="top" charoff="50">99.8 (73.2)</td><td align="center" valign="top" charoff="50">99.9 (50.9)</td><td align="center" valign="top" charoff="50">99.5 (46.7)</td><td align="center" valign="top" charoff="50">100 (33.3)</td><td align="center" valign="top" charoff="50">99.7 (35)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>I</italic>/<italic>σI</italic><xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">24.68 (1.46)</td><td align="center" valign="top" charoff="50">7.99 (0.89)</td><td align="center" valign="top" charoff="50">17.92 (1.85)</td><td align="center" valign="top" charoff="50">21.24 (1.07)</td><td align="center" valign="top" charoff="50">16.53 (1.41)</td><td align="center" valign="top" charoff="50">10.61 (0.97)</td><td align="center" valign="top" charoff="50">6.35 (1.00)</td><td align="center" valign="top" charoff="50">18.95 (0.92)</td><td align="center" valign="top" charoff="50">9.05 (0.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness<xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">99.9 (99.9)</td><td align="center" valign="top" charoff="50">99.6 (100)</td><td align="center" valign="top" charoff="50">99.7 (96.8)</td><td align="center" valign="top" charoff="50">99.8 (99.1)</td><td align="center" valign="top" charoff="50">99.8 (99.7)</td><td align="center" valign="top" charoff="50">99.7 (99.9)</td><td align="center" valign="top" charoff="50">99.4 (98.6)</td><td align="center" valign="top" charoff="50">99.6 (100)</td><td align="center" valign="top" charoff="50">99.1 (99.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy<xref ref-type="fn" rid="t1-fn2">†</xref></td><td align="center" valign="top" charoff="50">39.1 (39.8)</td><td align="center" valign="top" charoff="50">12.1 (14.3)</td><td align="center" valign="top" charoff="50">81.6 (65.2)</td><td align="center" valign="top" charoff="50">13.7 (13.7)</td><td align="center" valign="top" charoff="50">20.9 (19.1)</td><td align="center" valign="top" charoff="50">12.7 (13.5)</td><td align="center" valign="top" charoff="50">6.1 (6.5)</td><td align="center" valign="top" charoff="50">9.9 (10.4)</td><td align="center" valign="top" charoff="50">18.5 (18.2)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td><td> </td></tr><tr><td colspan="10" align="left" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution<xref ref-type="fn" rid="t1-fn1">*</xref> (Å)</td><td align="center" valign="top" charoff="50">46.4–3.0</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">84.5–3.7</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">49.2–3.6</td><td align="center" valign="top" charoff="50">49.1–4.5</td><td align="center" valign="top" charoff="50">49.9–7.2</td><td align="center" valign="top" charoff="50">50.0–8.4</td></tr><tr><td align="left" valign="top" charoff="50"> Reflections</td><td align="center" valign="top" charoff="50">16,928</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">40,647</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">41,799</td><td align="center" valign="top" charoff="50">23,340</td><td align="center" valign="top" charoff="50">14,046</td><td align="center" valign="top" charoff="50">12,111</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic>_work/<italic>R</italic>_free</td><td align="center" valign="top" charoff="50">0.20/0.24</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.35/0.38<xref ref-type="fn" rid="t1-fn3">‡</xref></td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.20/0.24</td><td align="center" valign="top" charoff="50">0.24/0.24</td><td align="center" valign="top" charoff="50">0.23/0.25</td><td align="center" valign="top" charoff="50">0.30/0.32</td></tr><tr><td align="left" valign="top" charoff="50"> Number of atoms</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">5,465</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">6,925</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">16,592</td><td align="center" valign="top" charoff="50">16,405</td><td align="center" valign="top" charoff="50">22,543</td><td align="center" valign="top" charoff="50">22,445</td></tr><tr><td align="left" valign="top" charoff="50">  Waters</td><td align="center" valign="top" charoff="50">43</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">7</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">5</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>B</italic>-factors</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  Protein</td><td align="center" valign="top" charoff="50">130</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">158</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">226</td><td align="center" valign="top" charoff="50">275</td><td align="center" valign="top" charoff="50">272</td><td align="center" valign="top" charoff="50">250</td></tr><tr><td align="left" valign="top" charoff="50">  Waters</td><td align="center" valign="top" charoff="50">84</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50">  Ligand/ion</td><td align="center" valign="top" charoff="50">90</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">189</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td></tr><tr><td align="left" valign="top" charoff="50"> R.m.s.d.</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td align="left" valign="top" charoff="50">  RMS (angles, °)</td><td align="center" valign="top" charoff="50">0.97</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.83</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">1.07</td><td align="center" valign="top" charoff="50">1.11</td><td align="center" valign="top" charoff="50">1.15</td><td align="center" valign="top" charoff="50">1.01</td></tr><tr><td align="left" valign="top" charoff="50">  RMS (bonds, Å)</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">—</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">0.01</td><td align="center" valign="top" charoff="50">0.01</td></tr></tbody></table> 54480   SceCD SceCD Thimerosal SceCD Eu HsaBT-CD HsaBT-CD Cd2+ CthCD-CTCter1 CthCD-CTCter2 CthCD-CT CthΔBCCP Data collection  Space group P43212 P43212 P43212 I4122 I4122 P212121 P212121 P31212 P6422  Cell dimensions                     a, b, c (Å) 110.86, 110.86, 131.12 111.22, 111.22, 131.49 108.65, 108.65, 127.36 267.27, 267.27, 210.61 267.67, 267.67, 210.46 97.66, 165.34, 219.23 100.17, 153.45, 249,24 295.02, 295.02, 189.52 462.20, 462.20, 204.64   α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 120 90, 90, 120  Resolution* (Å) 3.0 3.4 4.0 3.7 4.1 3.6 4.5 7.2 8.4  RMerge† 18.2 (389.6) 20.5 (306.1) 40.6 (327.0) 7.5 (400.9) 15 (730.5) 14.5 (384.5) 27.4 (225.6) 5.6 (302.6) 29.4 (381.7)  CC ½*† 100 (58.3) 99.9 (42.6) 99.9 (48.5) 100 (59.4) 99.8 (73.2) 99.9 (50.9) 99.5 (46.7) 100 (33.3) 99.7 (35)  I/σI† 24.68 (1.46) 7.99 (0.89) 17.92 (1.85) 21.24 (1.07) 16.53 (1.41) 10.61 (0.97) 6.35 (1.00) 18.95 (0.92) 9.05 (0.9)  Completeness† 99.9 (99.9) 99.6 (100) 99.7 (96.8) 99.8 (99.1) 99.8 (99.7) 99.7 (99.9) 99.4 (98.6) 99.6 (100) 99.1 (99.9)  Redundancy† 39.1 (39.8) 12.1 (14.3) 81.6 (65.2) 13.7 (13.7) 20.9 (19.1) 12.7 (13.5) 6.1 (6.5) 9.9 (10.4) 18.5 (18.2)                     Refinement  Resolution* (Å) 46.4–3.0     84.5–3.7   49.2–3.6 49.1–4.5 49.9–7.2 50.0–8.4  Reflections 16,928 — — 40,647 — 41,799 23,340 14,046 12,111  Rwork/Rfree 0.20/0.24 — — 0.35/0.38‡ — 0.20/0.24 0.24/0.24 0.23/0.25 0.30/0.32  Number of atoms                     Protein 5,465     6,925   16,592 16,405 22,543 22,445   Waters 43 — — — — — — — —   Ligand/ion 7 — — 5 — — — — —  B-factors                     Protein 130     158   226 275 272 250   Waters 84 — — — — — — — —   Ligand/ion 90 — — 189 — — — — —  R.m.s.d.                     RMS (angles, °) 0.97 — — 0.83 — 1.07 1.11 1.15 1.01   RMS (bonds, Å) 0.01 — — 0.01 — 0.01 0.01 0.01 0.01 t1.xml t1 TABLE table_footnote 56655 *Resolution cutoffs determined based on internal correlation significant at the 0.1% level as calculated by XDS. t1.xml t1 TABLE table_footnote 56768 †Highest-resolution shell is shown in parentheses. t1.xml t1 TABLE table_footnote 56821 ‡Modelled only as poly-alanine.