title: Structural basis of furan-amino acid recognition by a polyspecific aminoacyl-tRNA-synthetase and its genetic encoding in human cells.
abstract: The site-selective introduction of photo-crosslinking groups into proteins enables the discovery and mapping of weak and/or transient protein interactions with high spatiotemporal resolution, both in vitro and in vivo. We report the genetic encoding of a furan-based, photo-crosslinking amino acid in human cells; it can be activated with red light, thus offering high penetration depths in biological samples. This is achieved by activation of the amino acid and charging to its cognate tRNA by a pyrrolysyl-tRNA-synthetase (PylRS) mutant with broad polyspecificity. To gain insights into the recognition of this amino acid and to provide a rationale for its polyspecificity, we solved three crystal structures of the PylRS mutant: in its apo-form, in complex with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) and in complex with the AMP ester of the furan amino acid. These structures provide clues for the observed polyspecificity and represent a promising starting point for the engineering of PylRS mutants with further increased substrate scope.
authors.affiliation: Department of Chemistry, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz (Germany).
authors.forename: Moritz J
authors.initials: MJ
authors.lastname: Schmidt
authors.forename: Annemarie
authors.initials: A
authors.lastname: Weber
authors.forename: Moritz
authors.initials: M
authors.lastname: Pott
authors.forename: Wolfram
authors.initials: W
authors.lastname: Welte
authors.forename: Daniel
authors.initials: D
authors.lastname: Summerer
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005663:Furans / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D000596:Amino Acids; D005663:Furans; D000604:Amino Acyl-tRNA Synthetases; C039281:furan
keywords: X-ray structures; amino acids; crosslinking reactions; furans; protein modifications; pyrrolysyl tRNA synthetase