title: NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.
abstract: Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap-binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut-off host-cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot-and-mouth disease virus (FMDV) leader proteinase (Lb(pro)), human rhinovirus 2 (HRV2) 2A proteinase (2A(pro)) and coxsackievirus B4 (CVB4) 2A(pro) with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed (13)C/(15) N sequential backbone assignment of human eIF4GII residues 551-745 and examined their binding to murine eIF4E. eIF4GII551-745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain-like Lb(pro) only forms a stable complex with eIF4GII(551-745) in the presence of eIF4E, with KD values in the low nanomolar range; Lb(pro) contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin-like 2A(pro) from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K(D) values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut-off.
authors.affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, A-1030, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Aumayr
authors.affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, A-1030, Austria.
authors.forename: Sofiya
authors.initials: S
authors.lastname: Fedosyuk
authors.affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, A-1030, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Ruzicska
authors.affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, A-1030, Austria.
authors.forename: Carla
authors.initials: C
authors.lastname: Sousa-Blin
authors.affiliation: Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, Vienna, A-1030, Austria.
authors.forename: Georg
authors.initials: G
authors.lastname: Kontaxis
authors.affiliation: Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter, Dr. Bohr-Gasse 9/3, Vienna, A-1030, Austria.
authors.forename: Tim
authors.identifier: http://orcid.org/0000-0001-8865-5500
authors.initials: T
authors.lastname: Skern
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D054884:Host-Pathogen Interactions; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D010849:Picornaviridae / Q000737:chemistry / Q000201:enzymology* / Q000502:physiology; D011485:Protein Binding; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C491300:EIF4G2 protein, human; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D014764:Viral Proteins; C000601282:eIF4E protein, mouse; D010447:Peptide Hydrolases
keywords: control of protein synthesis; protein-protein interactions; substrate binding; viral proteases; virus-host interactions