title: Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination.
abstract: Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, N-21, W-11, Sapporo 001-0021, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kobashigawa
authors.forename: Tomohide
authors.initials: T
authors.lastname: Saio
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ushio
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Sekiguchi
authors.forename: Masashi
authors.initials: M
authors.lastname: Yokochi
authors.forename: Kenji
authors.initials: K
authors.lastname: Ogura
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D005470:Fluorometry / Q000379:methods*; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008280:Magnetics; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000378:metabolism; D044366:Transition Temperature
chemical_list: D028581:Lanthanoid Series Elements; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A