title: The "cap-binding slot" of an mRNA cap-binding protein: quantitative effects of aromatic side chain choice in the double-stacking sandwich with cap.
abstract: The N7-methylguanine portion of the mRNA cap structure interacts with cap-binding proteins via an unusual double-stacking arrangement in which the positively charged cap is sandwiched between two parallel-oriented aromatic protein side chains. Three-dimensional costructures of cap with two mRNA cap-binding proteins, namely, translational initiation factor eIF4E and VP39 (the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase), have heretofore been reported. Despite striking similarities between the two proteins in the double stack with the cap, the stack differs most notably in the species of stacked side chain donated by the protein. Whereas eIF4E employs two tryptophans, VP39 uses a tyrosine and a phenylalanine. Here, we have generated tryptophan substitutions in VP39. Tryptophan substitution was shown, crystallographically, not to disrupt the maintenance of a bona fide parallel stack. However, the single-tryptophan and double-tryptophan substitutions were associated with increased affinity for cap nucleoside by factors of 10 and 50, respectively. VP39 interacted more strongly with a true substrate (containing portions of RNA downstream of the cap in addition to the cap itself) than with isolated cap nucleoside, by several orders of magnitude. VP39 mutants with tryptophan substitution at position 180 exhibited apparent defects in substrate catalytic rate during the first turnover cycle, indicating the possibility of an exquisite sensitivity of the catalytic center to subtle changes in substrate position brought about by alterations in the cap-binding slot. The X-ray structure of VP39 with a genuine nucleobase analogue of N7-methylguanosine, namely, N7,9-dimethylguanine, indicated that the N7-methylguanosine rotational orientation within the stack is a property of the cap nucleobase itself.
authors.affiliation: Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Hu
authors.forename: Akihiro
authors.initials: A
authors.lastname: Oguro
authors.forename: Changzheng
authors.initials: C
authors.lastname: Li
authors.forename: Paul D
authors.initials: PD
authors.lastname: Gershon
authors.forename: Florante A
authors.initials: FA
authors.lastname: Quiocho
mesh_terms: D018360:Crystallography, X-Ray; D039561:Eukaryotic Initiation Factor-4E; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity / Q000235:genetics; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012996:Solutions; C088682:VP39 protein, Vaccinia virus; D014764:Viral Proteins; D006151:Guanosine; C016578:7-methylguanosine; D008780:Methyltransferases; C055099:mRNA (nucleoside-O(2'))-methyltransferase