title: Functional conformations for pyruvate carboxylase during catalysis explored by cryoelectron microscopy.
abstract: The tetrameric enzyme pyruvate carboxylase (PC), a biotin-dependent carboxylase, produces oxaloacetate by two consecutive reactions that take place in distant active sites. Previous crystal structures revealed two different configurations for PC tetramers, the so-called symmetric and asymmetric, which were understood as characteristic molecular architectures for PC from different organisms. We have analyzed PC samples from Staphylococcus aureus while the enzyme generates oxaloacetate, expecting PC tetramers to display the conformational landscape relevant for its functioning. Using cryoelectron microscopy (cryo-EM) and sorting techniques, we detect previously defined symmetric and asymmetric architectures, demonstrating that PC maps both arrangements by large conformational changes. Furthermore, we observe that each configuration is coupled to one of the two consecutive enzymatic reactions. The findings describe the structural transitions relevant for the allosteric control of the multifunctional PC and demonstrate that by cryo-EM and classification, we can characterize freely working macromolecules.
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, 48160 Derio, Spain.
authors.forename: Gorka
authors.initials: G
authors.lastname: Lasso
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Linda P C
authors.initials: LP
authors.lastname: Yu
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, 48160 Derio, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Gil
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, 48160 Derio, Spain.
authors.forename: Melisa
authors.initials: M
authors.lastname: Lázaro
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.affiliation: Structural Biology Unit, Center for Cooperative Research in Biosciences, CIC bioGUNE, 48160 Derio, Spain; Department of Biochemistry and Molecular Biology, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain. Electronic address: mvalle@cicbiogune.es.
authors.forename: Mikel
authors.initials: M
authors.lastname: Valle
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002384:Catalysis; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011766:Pyruvate Carboxylase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D011766:Pyruvate Carboxylase