title: Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes.
abstract: The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.
authors.affiliation: Department of Cell Biology, Wenner-Gren Institute, University of Stockholm, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Nilsson
authors.forename: O
authors.initials: O
authors.lastname: Nygård
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014357:Trypsin
chemical_list: D000345:Affinity Labels; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D006160:Guanosine Triphosphate; D014357:Trypsin