title: Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity.
abstract: Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Yates
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Durrant
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Sophie
authors.initials: S
authors.lastname: Fleurdépine
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Karl
authors.initials: K
authors.lastname: Harlos
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK gilbert@strubi.ox.ac.uk.
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Norbury
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK gilbert@strubi.ox.ac.uk.
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012399:Rotation; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D012331:RNA, Fungal; D029702:Schizosaccharomyces pombe Proteins; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; C520554:Cid1 protein, S pombe; D014544:Uridine Triphosphate