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• W2466598902 abstract "•Crystal structures of core domains of a single-chain bacterial carboxylase•Analysis of large-scale domain motions in the 680-kDa carboxylase•Hybrid model integrating crystallographic and solution scattering data•Paradigm for the dynamic architecture of carrier protein-based multienzymes Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes. Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes. Biotin-dependent carboxylases are ubiquitous enzymes catalyzing ATP-dependent carboxylation in fatty acid, carbohydrate, and amino acid metabolism, as well as in urea utilization and microbial polyketide biosynthesis (Cronan and Waldrop, 2002Cronan Jr., J.E. Waldrop G.L. Multi-subunit acetyl-CoA carboxylases.Prog. Lipid Res. 2002; 41: 407-435Crossref PubMed Scopus (330) Google Scholar, Jitrapakdee et al., 2008Jitrapakdee S. St Maurice M. Rayment I. Cleland W.W. Wallace J.C. Attwood P.V. Structure, mechanism and regulation of pyruvate carboxylase.Biochem. J. 2008; 413: 369-387Crossref PubMed Scopus (298) Google Scholar, Tong, 2005Tong L. Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery.Cell Mol. Life Sci. 2005; 62: 1784-1803Crossref PubMed Scopus (366) Google Scholar, Wakil et al., 1983Wakil S.J. Stoops J.K. Joshi V.C. Fatty acid synthesis and its regulation.Annu. Rev. Biochem. 1983; 52: 537-579Crossref PubMed Google Scholar, Zhang et al., 2010Zhang H. Boghigian B.A. Pfeifer B.A. Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli.Biotechnol. Bioeng. 2010; 105: 567-573Crossref PubMed Scopus (54) Google Scholar). Based on their substrate specificity, biotin-dependent carboxylases are classified into three families: urea carboxylases (UCs), pyruvate carboxylases (PCs), and the general family of acyl-coenzyme A (CoA) carboxylases (aCCs) (Fan et al., 2012Fan C. Chou C.Y. Tong L. Xiang S. Crystal structure of urea carboxylase provides insights into the carboxyltransfer reaction.J. Biol. Chem. 2012; 287: 9389-9398Crossref PubMed Scopus (30) Google Scholar, Maurice et al., 2007Maurice M.S. Reinhardt L. Surinya K.H. Attwood P.V. Wallace J.C. Cleland W.W. Rayment I. Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme.Science. 2007; 317: 1076-1079Crossref PubMed Scopus (106) Google Scholar, Mobley and Hausinger, 1989Mobley H.L. Hausinger R.P. Microbial ureases: significance, regulation, and molecular characterization.Microbiol. Rev. 1989; 53: 85-108Crossref PubMed Google Scholar). The aCC family includes acetyl-CoA carboxylase (ACC), which catalyzes the key committed step in fatty acid biosynthesis, propionyl-CoA carboxylase (PCC), and 3-methylcrotonyl-CoA carboxylase (MCC) (Cronan and Waldrop, 2002Cronan Jr., J.E. Waldrop G.L. Multi-subunit acetyl-CoA carboxylases.Prog. Lipid Res. 2002; 41: 407-435Crossref PubMed Scopus (330) Google Scholar, Fall and Hector, 1977Fall R.R. Hector M.L. Acyl-coenzyme A carboxylases. Homologous 3-methylcrotonyl-CoA and geranyl-CoA carboxylases from Pseudomonas citronellolis.Biochemistry. 1977; 16: 4000-4005Crossref PubMed Scopus (29) Google Scholar, Huang et al., 2010Huang C.S. Sadre-Bazzaz K. Shen Y. Deng B. Zhou Z.H. Tong L. Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase.Nature. 2010; 466: 1001-1005Crossref PubMed Scopus (69) Google Scholar, Huang et al., 2011Huang C.S. Ge P. Zhou Z.H. Tong L. An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase.Nature. 2011; 481: 219-223Crossref PubMed Scopus (34) Google Scholar, Rodriguez et al., 2001Rodriguez E. Banchio C. Diacovich L. Bibb M.J. Gramajo H. Role of an essential acyl coenzyme a carboxylase in the primary and secondary metabolism of Streptomyces coelicolor.Appl. Environ. Microbiol. 2001; 67: 4166-4176Crossref PubMed Scopus (62) Google Scholar). Despite their diverse substrate specificities, all biotin-dependent carboxylases share a common enzymatic mechanism and domain organization. Carboxylation is carried out in two half-reactions and involves three functional components. First, a biotin carboxylase (BC) catalyzes the ATP-dependent carboxylation of a biotin cofactor, which is covalently linked to a conserved lysine of the biotin carboxyl carrier protein (BCCP). Then BCCP translocates to the carboxyl transferase (CT), where the carboxyl group of the carboxybiotin intermediate is transferred to the respective substrate (Attwood and Wallace, 2002Attwood P.V. Wallace J.C. Chemical and catalytic mechanisms of carboxyl transfer reactions in biotin-dependent enzymes.Acc. Chem. Res. 2002; 35: 113-120Crossref PubMed Scopus (99) Google Scholar, Knowles, 1989Knowles J.R. The mechanism of biotin-dependent enzymes.Annu. Rev. Biochem. 1989; 58: 195-221Crossref PubMed Scopus (358) Google Scholar). In this reaction scheme, the BC and BCCP are conserved components, while the CT varies in active-site structure depending on the substrate. The structural organization differs vastly between various aCCs, despite their conserved reaction logic. They either occur as multisubunit enzymes, where the enzymatic functions are provided by distinct protein subunits, or as single-chain multienzymes, which integrate all enzymatic domains into one polypeptide chain (Figure 1A ). The group of multisubunit aCCs comprises most prokaryotic forms, which consist of two, three, or four subunits as exemplified by Streptomyces coelicolor PCC, Metallosphaera sedula ACC, or Escherichia coli ACC, respectively, as well as the eukaryotic PCC and MCC. Single-chain aCCs are represented by eukaryotic ACCs, which comprise a large, non-catalytic central domain in addition to the canonical enzymatic domains. Strikingly different oligomeric assemblies are observed for aCCs. Bacterial PCC and MCC are hetero-dodecamers with (BC-BCCP)6(CT)6 stoichiometry (Huang et al., 2010Huang C.S. Sadre-Bazzaz K. Shen Y. Deng B. Zhou Z.H. Tong L. Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase.Nature. 2010; 466: 1001-1005Crossref PubMed Scopus (69) Google Scholar, Huang et al., 2011Huang C.S. Ge P. Zhou Z.H. Tong L. An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase.Nature. 2011; 481: 219-223Crossref PubMed Scopus (34) Google Scholar). Bacterial ACCs have a (BC)2(BCCP)4(CTαCTβ)2 stoichiometry (Tong, 2012Tong L. Structure and function of biotin-dependent carboxylases.Cell Mol. Life Sci. 2012; 70: 863-891Crossref PubMed Scopus (244) Google Scholar), and the archaeal ACC holoenzyme forms a (BC)4(BCCP)4(CT)4 hetero-dodecamer (Hügler et al., 2003Hügler M. Krieger R.S. Jahn M. Fuchs G. Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula.Eur. J. Biochem. 2003; 270: 736-744Crossref PubMed Scopus (87) Google Scholar). Eukaryotic ACCs function as dimers or higher oligomeric filaments (Hunkeler et al., 2016Hunkeler M. Stuttfeld E. Hagmann A. Imseng S. Maier T. The dynamic organization of fungal acetyl-CoA carboxylase.Nat. Commun. 2016; 7: 1-11Crossref Scopus (31) Google Scholar, Kim et al., 2010Kim C.-W. Moon Y.-A. Park S.W. Cheng D. Kwon H.J. Horton J.D. Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis.Proc. Natl. Acad. Sci. USA. 2010; 107: 9626-9631Crossref PubMed Scopus (92) Google Scholar, Lee et al., 2008Lee C.-K. Cheong H.-K. Ryu K.-S. Lee J.I. Lee W. Jeon Y.H. Cheong C. Biotinoyl domain of human acetyl-CoA carboxylase: structural insights into the carboxyl transfer mechanism.Proteins. 2008; 72: 613-624Crossref PubMed Scopus (18) Google Scholar, Wei and Tong, 2015Wei J. Tong L. Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer.Nature. 2015; 526: 723-727Crossref PubMed Scopus (50) Google Scholar). Recently, a group of single-chain aCCs of as yet unknown substrate specificity was identified in bacteria. These enzymes, denoted YCCs, encompass all functional domains in a single polypeptide chain of ∼1,200 amino acids. They share the domain order with eukaryotic ACCs, but lack the large central domain (Figure 1A). YCCs occur in a diverse set of unrelated bacterial species, including Pseudomonas aeruginosa, Cupriavidus metallidurans, and Deinococcus radiodurans. For most YCCs, neither the substrates nor their physiological function or structure have been identified. The only exceptions are Mycobacterium avium LCC (Map LCC) and Rhodopseudomonas palustris LCC (Rpa LCC), for which a crystal structure and an initial substrate characterization, respectively, have been obtained recently (Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar). Based on high-resolution crystal structures of the CT and BC subunits and solution structural data, we report a hybrid model of the D. radiodurans YCC (Dra YCC), which provides insights into active-site architecture, domain interactions, and the dynamic organization of a complex YCC multienzyme. Dra YCC was overexpressed in Sf21 insect cells and purified in biotinylated form with a yield of ∼30 mg per liter of culture. It forms a hexamer of ∼680 kDa in solution, as confirmed by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) (Figure S1A). SEC and SDS-PAGE analysis of the protein confirmed homogeneity and purity of the sample (Figures S1B and S1C). We employed a hybrid approach to determine high-resolution crystal structures of the individual enzymatic domains in combination with analysis of the overall assembly structure and flexibility in solution by small-angle X-ray scattering (SAXS) and electron microscopy (EM). We overexpressed the CT domain of Dra YCC (amino acids 556–1,091) in Sf21 cells. The protein forms a hexamer of ∼330 kDa in solution based on SEC-MALS analysis (Figure S1D). The CT domain was crystallized in space group P212121 and its structure was determined by molecular replacement using the transcarboxylase 12S subunit (Hall, 2003Hall P.R. Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core.EMBO J. 2003; 22: 2334-2347Crossref PubMed Scopus (43) Google Scholar) as search model. The final model comprising residues 573–1,091 was refined to Rwork/Rfree at 20.2%/24.1% at 2.4-Å resolution (Table 1). The CT domain consists of two subdomains, the N lobe (residues 554–832) and the C lobe (residues 833–1,091). Consistent with the oligomeric state in solution, the CT domain forms a hexameric ring with D3 symmetry, consisting of a trimer of dimers with an outer diameter of ∼140 Å and a height of ∼67 Å (Figures 1B and 1C). Dimerization of CT is mediated by head-to-tail interaction of two protomers with an interface area of ∼3900 Å2. Both subdomains contribute to trimerization of CT dimers via an interface area of ∼2800 Å2 per dimer.Table 1Crystallographic Data Collection and Refinement Statistics for Dra YCC CT and BCDra YCC CTDra YCC BCWavelength (Å)1.000031.0000Resolution range (Å)69.5–2.49 (2.58–2.49)75.74–1.70 (1.8–1.70)Space groupP 21 21 21P 65Unit cell (​Å)111.47, 149.57, 189.3138.02, 138.02, 97.81α, β, γ (°)90, 90, 9090, 90, 120Total reflections1,497,948 (237,650)1,181,171 (180642)Unique reflections110,205 (17,213)116,236 (18,405)Multiplicity13.6 (13.8)10.1 (9.8)Completeness (%)99.64 (97.4)99.87 (97.8)Mean I/σ13.34 (1.90)15.61 (1.18)Wilson B factor (Å2)40.7832.17Twin operator/twin fraction (%)–k,h,-l/30.6Rmeas0.220.10CC1/20.997 (0.676)0.999 (0.461)Rwork0.2020.149Rfree0.2410.184No. of atoms46,3267,346Macromolecules45,5646,529Ligands8028Waters682789Protein residues3,089856RMSD (angles)0.832.46RMSD (bonds)0.00340.0262Ramachandran favored (%)96.396.3Ramachandran outliers (%)0.910.71Clashscore1.93.3Values in parentheses correspond to the highest-resolution shell. Open table in a new tab Values in parentheses correspond to the highest-resolution shell. A comparison of the Dra YCC and Map LCC CT structures (Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar) shows conservation of oligomeric interaction and overall assembly shape. However, a major difference is observed in the intersubunit dimer interface: Dra YCC features a 15-amino-acid C-terminal extension (Figure 1D), which enlarges the dimer interface by ∼700 Å2 relative to Map LCC. The C-terminal extension protrudes into the dimer interface from the inside of the hexameric ring, while Map LCC already terminates at the end of a conserved helix (Figure S2A). Based on sequence alignment, a C-terminal extension of ∼10–20 amino acids with a highly conserved aspartate-X-tryptophan (DXW) motif at the very C terminus is present in 20 out of 29 YCC sequences (Figure S2B). The C-terminal DXW motif mediates Dra YCC intersubunit interactions: the motif's aspartate 1,089 and the C-terminal carboxyl group are both engaged in bidentate interactions with the conserved arginine 957 of the neighboring protomer (Figure 1E). Arginine 957 is also found in Map LCC, which lacks a C-terminal extension, and there forms a salt bridge to aspartate 919 in a neighboring helix from the same subunit. The tryptophan of the DXW motif stacks with histidine 955, which is conserved in all but one of the examined YCCs. C-Terminal core fold extensions of similar length and with distinct conserved C-terminal motifs are also present in the two other families of structurally characterized hexameric CT domains, namely PCCs and MCCs (Figures S2C and S2D) (Arabolaza et al., 2010Arabolaza A. Shillito M.E. Lin T.-W. Diacovich L. Melgar M. Pham H. Amick D. Gramajo H. Tsai S.-C. Crystal structures and mutational analyses of acyl-CoA carboxylase β subunit of Streptomyces coelicolor.Biochemistry. 2010; 49: 7367-7376Crossref PubMed Scopus (34) Google Scholar, Huang et al., 2011Huang C.S. Ge P. Zhou Z.H. Tong L. An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase.Nature. 2011; 481: 219-223Crossref PubMed Scopus (34) Google Scholar). In PCC, these extensions extend into the same region of the hexameric CT assembly and terminate at the equivalent position of the Dra YCC C-terminal extension. However, in PCC the extensions are not crossed over (Figure S2C). In MCC, which is characterized by a domain-swapped organization of the CT N and C lobes relative to YCC and PCC, the C-terminal extensions adopt a completely different topology and protrude into the interface responsible for dimer trimerization (Figure S2D) (Huang et al., 2011Huang C.S. Ge P. Zhou Z.H. Tong L. An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase.Nature. 2011; 481: 219-223Crossref PubMed Scopus (34) Google Scholar). As in YCC and PCC, the extension only spans about two-thirds of the interface. Overall, C-terminal core fold extensions are observed in all structurally characterized hexameric CT domains, except for the Map LCC-like subclass of YCCs. In these systems, the C-terminal extensions are located in intersubunit or interdomain interfaces. However, their topology and the interactions of their distinct conserved C-terminal motifs are strikingly divergent. The CT active site is formed at the dimer interface between the N lobe of one protomer and the C lobe of its dimer partner (Figure 1C). It consists of a biotin- and substrate-specific binding pocket. The substrate binding pocket of Dra YCC is an elongated and narrow cleft of ∼20 Å length (Figure 2A ) and a volume of ∼2000 Å3, which is lined by conserved residues and sufficient in length to enclose a C16 acyl chain with possible space for accommodation of longer or branched substrates. The binding groove is much more extended than in ACCs with their small acetyl substrate, e.g., Mycobacterium tuberculosis ACC CT (Reddy et al., 2014Reddy M.C.M. Breda A. Bruning J.B. Sherekar M. Valluru S. Thurman C. Ehrenfeld H. Sacchettini J.C. Structure, activity, and inhibition of the carboxyltransferase-subunit of acetyl coenzyme a carboxylase (AccD6) from Mycobacterium tuberculosis.Antimicrob. Agents Chemother. 2014; 58: 6122-6132Crossref PubMed Scopus (16) Google Scholar) (Figures 2B and S3A). The active-site region of Dra YCC is rather conserved at the sequence level to Rpa LCC, which is active toward C2 to C16 substrates, but with a preference for long-chain acyl chain substrates (Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar). However, in the crystal structure of the related Map LCC, the substrate binding cleft is occluded by a loop formed by residues 715–732 (Figures 2B and S3B), such that a requirement for conformational changes during substrate accommodation has been suggested (Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar). The corresponding loop glycine 716-proline 732 in Dra YCC adopts a different conformation, which results in an opening of the substrate binding groove. Dra YCC may thus represent a substrate binding competent form of the active site. In Map LCC, a helix spanning residues 781–787 occludes the CoA binding region of the active site (Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar). Although the positioning of the corresponding helix (residues 781–788) is well conserved in the Dra YCC CT structure, already a slight shift of the CoA moiety relative to its position observed in the crystal structure of MCC would be sufficient for accommodating CoA in an equivalent pocket also in Dra YCC CT. The respective regions are not constrained by crystal contacts in either the Dra YCC CT or Map LCC CT crystal structures. A helical hairpin flap (amino acids 1,005–1,046) with increased flexibility is positioned above the active site of Dra YCC. In the structure of the homologous P. aeruginosa MCC CT domain in complex with CoA, the corresponding flap acts as a lid helix by closing down onto the bound CoA moiety (Huang et al., 2011Huang C.S. Ge P. Zhou Z.H. Tong L. An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase.Nature. 2011; 481: 219-223Crossref PubMed Scopus (34) Google Scholar), suggesting a similar role in CoA and substrate accommodation also in Dra YCC (Figure 1C). Initial expression screening failed to yield a solubly expressed construct of the Dra YCC BC domain. However, a stable BC domain fragment was obtained by limited proteolysis of full-length Dra YCC. Initial crystals of the fragment were affected by merohedral twinning with a twin fraction close to 50%, which precluded structure determination. Through extensive screening, a crystal with a twin fraction of ∼30% in space group P65 with two molecules in the asymmetric unit was obtained and the Dra YCC BC domain structure solved using molecular replacement with the BC domain of PC (Kondo et al., 2007Kondo S. Nakajima Y. Sugio S. Sueda S. Islam M.N. Kondo H. Structure of the biotin carboxylase domain of pyruvate carboxylase from Bacillus thermodenitrificans.Acta Crystallogr. D Biol. Crystallogr. 2007; 63: 885-890Crossref PubMed Scopus (11) Google Scholar). The final model consists of two almost identical monomers (root-mean-square deviation [RMSD] of 0.14 Å) (Figure S4A) including residues 1–464 and was refined to Rwork/Rfree of 14.9%/18.4% at 1.7-Å resolution using twin refinement in REFMAC5 (Vagin et al., 2004Vagin A.A. Steiner R.A. Lebedev A.A. Potterton L. McNicholas S. Long F. Murshudov G.N. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2184-2195Crossref PubMed Scopus (1063) Google Scholar). The BC domain consists of the rigid A and C subdomains and the small interspersed B subdomain (Figure 3A ) (Waldrop et al., 2012Waldrop G.L. Holden H.M. Maurice M.S. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.Protein Sci. 2012; 21: 1597-1619Crossref PubMed Scopus (64) Google Scholar). The B subdomain is flexibly tethered by two irregular peptide linkers and exhibits increased disorder in the crystal (Figure S4B). Based on homology to the BC domains of E. coli or Haemophilus influenzae ACC, this B subdomain may undergo a hinge-bending motion during substrate binding to act as a lid to the BC active site (Broussard et al., 2015Broussard T. Pakhomova S. Neau D.B. Bonnot R. Waldrop G.L. Structural analysis of substrate, reaction intermediate and product binding in Haemophilus influenzae biotin carboxylase.Biochemistry. 2015; 54: 3860-3870Crossref PubMed Scopus (12) Google Scholar, Thoden et al., 2000Thoden J.B. Blanchard C.Z. Holden H.M. Waldrop G.L. Movement of the biotin carboxylase B-domain as a result of ATP binding.J. Biol. Chem. 2000; 275: 16183-16190Crossref PubMed Scopus (96) Google Scholar). Particularly the highly conserved glycine-rich loop (lysine 157-methionine 167) in the B subdomain might be involved in direct ligand contacts, with lysine 157 presumably interacting with one of the α-phosphoryl oxygens of MgATP (Figure 3A). The BC active site is located at the interface of the A and C subdomains. Active-site residues are well conserved between YCC, ACC (Waldrop et al., 2012Waldrop G.L. Holden H.M. Maurice M.S. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.Protein Sci. 2012; 21: 1597-1619Crossref PubMed Scopus (64) Google Scholar), and PCC BC (Huang et al., 2010Huang C.S. Sadre-Bazzaz K. Shen Y. Deng B. Zhou Z.H. Tong L. Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase.Nature. 2010; 466: 1001-1005Crossref PubMed Scopus (69) Google Scholar) (Figure 3B). Structural homology suggests that residues glutamic acid 273 and glutamic acid 286 in Dra YCC BC coordinate magnesium ions, which are presumably involved in binding of the ATP substrate. Arginine 290, glutamic acid 294, and arginine 342 are directly implicated in the binding of biotin and ATP (Broussard et al., 2015Broussard T. Pakhomova S. Neau D.B. Bonnot R. Waldrop G.L. Structural analysis of substrate, reaction intermediate and product binding in Haemophilus influenzae biotin carboxylase.Biochemistry. 2015; 54: 3860-3870Crossref PubMed Scopus (12) Google Scholar, Huang et al., 2010Huang C.S. Sadre-Bazzaz K. Shen Y. Deng B. Zhou Z.H. Tong L. Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase.Nature. 2010; 466: 1001-1005Crossref PubMed Scopus (69) Google Scholar, Waldrop et al., 2012Waldrop G.L. Holden H.M. Maurice M.S. The enzymes of biotin dependent CO2 metabolism: what structures reveal about their reaction mechanisms.Protein Sci. 2012; 21: 1597-1619Crossref PubMed Scopus (64) Google Scholar). Carboxybiotin-interacting residues have been identified in the crystal structure of the H. influenzae ACC BC (Broussard et al., 2015Broussard T. Pakhomova S. Neau D.B. Bonnot R. Waldrop G.L. Structural analysis of substrate, reaction intermediate and product binding in Haemophilus influenzae biotin carboxylase.Biochemistry. 2015; 54: 3860-3870Crossref PubMed Scopus (12) Google Scholar) and are structurally conserved in Dra YCC (lysine 235, arginine 290, and arginine 342; Figure 3B), in agreement with a generally conserved BC mechanism. The BC domains form dimers with an interface area of ∼900 Å2 and a maximum extent of 100 Å, analogous to the E. coli and H. influenzae ACC BC as well as the BC domain of the Map LCC (Broussard et al., 2015Broussard T. Pakhomova S. Neau D.B. Bonnot R. Waldrop G.L. Structural analysis of substrate, reaction intermediate and product binding in Haemophilus influenzae biotin carboxylase.Biochemistry. 2015; 54: 3860-3870Crossref PubMed Scopus (12) Google Scholar, Chou et al., 2008Chou C.Y. Yu L.P.C. Tong L. Crystal structure of biotin carboxylase in complex with substrates and implications for its catalytic mechanism.J. Biol. Chem. 2008; 284: 11690-11697Crossref Scopus (53) Google Scholar, Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar). Contrastingly, the BC domains of eukaryotic ACC are monomers in isolation, but may form a permanent or transient dimer in context of the full-length ACC multienzyme (Cho et al., 2007Cho Y.S. Lee J.I. Shin D. Kim H.T. Cheon Y.H. Seo C.I. Kim Y.E. Hyun Y.-L. Lee Y.S. Sugiyama K. et al.Crystal structure of the biotin carboxylase domain of human acetyl-CoA carboxylase 2.Proteins. 2007; 70: 268-272Crossref Scopus (13) Google Scholar, Hunkeler et al., 2016Hunkeler M. Stuttfeld E. Hagmann A. Imseng S. Maier T. The dynamic organization of fungal acetyl-CoA carboxylase.Nat. Commun. 2016; 7: 1-11Crossref Scopus (31) Google Scholar, Wei and Tong, 2015Wei J. Tong L. Crystal structure of the 500-kDa yeast acetyl-CoA carboxylase holoenzyme dimer.Nature. 2015; 526: 723-727Crossref PubMed Scopus (50) Google Scholar). To gain insight into the overall Dra YCC assembly, we employed negative-stain EM. Dra YCC particles are characterized by a central triangular shape, which corresponds to the hexameric CT domain (Figure 4A ). 9,477 particles were picked and used for the generation of 24 2D class averages (Figure S5). Sixteen of these 24 classes represent top views of the CT hexamer with laterally positioned dimeric BC domains (Figure 4B). Only one class provides a side view along a two-fold symmetry axis; the remaining classes are projections showing the CT domain tilted to ∼45°. Projections of the CT crystal structure correspond well to the respective class averages (Figure 4C). While the CT hexamer is preserved in all classes, significant variability is observed in positioning of the BC domains. To further analyze BC positioning, we measured the pixel distances between the center of the CT hexamer and the respective BC dimers in particles of top-view classes as an approximation of CT-BC distance. ∼1,600 distance measurements were sorted into classes of 5-Å increments (Figure 4D). The resulting distance histogram demonstrates that BC dimer positioning is highly variable and covers the total range from less than 80 Å, which corresponds to the BC dimer resting on the CT ring as observed in the Map LCC crystal structure (Tran et al., 2015Tran T.H. Hsiao Y.-S. Jo J. Chou C.-Y. Dietrich L.E.P. Walz T. Tong L. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.Nature. 2015; 518: 120-123Crossref PubMed Scopus (25) Google Scholar), to more than 135 Å from the center of the CT ring, which roughly corresponds to maximally extended interdomain linker conformation. Only about 12% of all BC domains were detected in close proximity with the CT domains at distances to the CT center of <85 Å. The distance distribution has a maximum at an intermediate distance of 105 Å. However, due to measurement in projection images, this value probably still underestimates the rea" @default.
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• W2466598902 date "2016-08-01" @default.
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• W2466598902 title "Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans" @default.
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• W2466598902 doi "https://doi.org/10.1016/j.str.2016.06.001" @default.
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