title: NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.
abstract: G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of (15)N-Trp Gbetagamma identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GbetaW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GbetaW99 is highly mobile on the nanosecond timescale in comparison with other Gbeta tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GbetaW99 and GbetaW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G(alpha)(i1)-GDP to Gbetagamma had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.
authors.affiliation: Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain. Alan_Smrcka@urmc.rochester.edu
authors.forename: Alan V
authors.initials: AV
authors.lastname: Smrcka
authors.forename: Nessim
authors.initials: N
authors.lastname: Kichik
authors.forename: Teresa
authors.initials: T
authors.lastname: Tarragó
authors.forename: Michael
authors.initials: M
authors.lastname: Burroughs
authors.forename: Min-Sun
authors.initials: MS
authors.lastname: Park
authors.forename: Nathan K
authors.initials: NK
authors.lastname: Itoga
authors.forename: Harry A
authors.initials: HA
authors.lastname: Stern
authors.forename: Barry M
authors.initials: BM
authors.lastname: Willardson
authors.forename: Ernest
authors.initials: E
authors.lastname: Giralt
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D011789:Quantum Theory; D014364:Tryptophan / Q000737:chemistry
chemical_list: D008024:Ligands; D009587:Nitrogen Isotopes; D010455:Peptides; D021122:Protein Subunits; D006153:Guanosine Diphosphate; C011265:guanosine 5'-(beta,gamma-methylene)triphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D019204:GTP-Binding Proteins