title: Towards a structural understanding of the smallest known oncoprotein: investigation of the bovine papillomavirus E5 protein using solution-state NMR.
abstract: The homodimeric E5 protein from bovine papillomavirus activates the platelet-derived growth factor β receptor through transmembrane (TM) helix-helix interactions leading to uncontrolled cell growth. Detailed structural information for the E5 dimer is essential if we are to uncover its unique mechanism of action. In vivo mutagenesis has been used to identify residues in the TM domain critical for dimerization, and we previously reported that a truncated synthetic E5 peptide forms dimers via TM domain interactions. Here we extend this work with the first application of high-resolution solution-state NMR to the study of the E5 TM domain in SDS micelles. Using selectively 15N-labelled peptides, we first probe sample homogeneity revealing two predominate species, which we interpret to be monomer and dimer. The equilibrium between the two states is shown to be dependent on detergent concentration, revealed by intensity shifts between two sets of peaks in 15N-(1)H HSQC experiments, highlighting the importance of sample preparation when working with these types of proteins. This information is used to estimate a free energy of association (ΔGx°=-3.05 kcal mol(-1)) for the dimerization of E5 in SDS micelles. In addition, chemical shift changes have been observed that indicate a more pronounced change in chemical environment for those residues expected to be at the dimer interface in vivo versus those that are not. Thus we are able to demonstrate our in vitro dimer is comparable to that defined in vivo, validating the biological significance of our synthetic peptide and providing a solid foundation upon which to base further structural studies. Using detergent concentration to modulate oligomeric state and map interfacial residues by NMR could prove useful in the study of other homo-oligomeric transmembrane proteins.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Gavin
authors.initials: G
authors.lastname: King
authors.forename: Joanne
authors.initials: J
authors.lastname: Oates
authors.forename: Dharmesh
authors.initials: D
authors.lastname: Patel
authors.forename: Hugo A
authors.initials: HA
authors.lastname: van den Berg
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dixon
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D052688:Bovine papillomavirus 1 / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012996:Solutions; D013816:Thermodynamics
chemical_list: D009856:Oncogene Proteins, Viral; D012996:Solutions; C054304:oncogene protein E5, Bovine papillomavirus type 1; D012967:Sodium Dodecyl Sulfate; D010649:Phenylalanine; D007930:Leucine; D000409:Alanine