title: Probing riboswitch-ligand interactions using thiamine pyrophosphate analogues.
abstract: The Escherichia coli thiM riboswitch forms specific contacts with its natural ligand, thiamine pyrophosphate (TPP or thiamine diphosphate), allowing it to generate not only nanomolar binding affinity, but also a high degree of discrimination against similar small molecules. A range of synthetic TPP analogues have been used to probe each of the riboswitch-ligand interactions. The results show that the pyrimidine-sensing helix of thiM is exquisitely tuned to select for TPP by recognising the H-bonding donor and acceptors around its aminopyrimidine ring and also by forming π-stacking interactions that may be sensitive to the electronics of the ring. The central thiazolium ring of TPP appears to be more important for ligand recognition than previously thought. It may contribute to binding via long-range electrostatic interactions and/or by exerting an electron withdrawing effect on the pyrimidine ring, allowing its presence to be sensed indirectly and thereby allowing discrimination between thiamine (and its phosphate esters) and other aminopyrimidines found in vivo. The pyrophosphate moiety is essential for submicromolar binding affinity, but unexpectedly, it does not appear to be strictly necessary for modulation of gene expression.
authors.affiliation: University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Liuhong
authors.initials: L
authors.lastname: Chen
authors.forename: Elena
authors.initials: E
authors.lastname: Cressina
authors.forename: Neil
authors.initials: N
authors.lastname: Dixon
authors.forename: Karl
authors.initials: K
authors.lastname: Erixon
authors.forename: Kwasi
authors.initials: K
authors.lastname: Agyei-Owusu
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
authors.forename: Alison G
authors.initials: AG
authors.lastname: Smith
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.forename: Finian J
authors.initials: FJ
authors.lastname: Leeper
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression / Q000187:drug effects; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011743:Pyrimidines / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058928:Riboswitch / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D011743:Pyrimidines; D012329:RNA, Bacterial; D058928:Riboswitch; C030986:pyrimidine; D013835:Thiamine Pyrophosphate