ABSTRACT
The first low resolution solution structure of the soluble domain of subunit b (b (22-156)) of the Escherichia coli F(1)F(O) ATPsynthase was determined from small-angle X-ray scattering data. The dimeric protein has a boomerang-like shape with a total length of 16.2 +/- 0.3 nm. Fluorescence correlation spectroscopy (FCS) shows that the protein binds effectively to the subunit delta, confirming their described neighborhood. Using the recombinant C-terminal domain (delta(91-177)) of subunit delta and the C-terminal peptides of subunit b, b (120-140) and b (140-156), FCS titration experiments were performed to assign the segments involved in delta-b assembly. These data identify the very C-terminal tail b (140-156) to interact with delta(91-177). The novel 3D structure of this peptide has been determined by NMR spectroscopy. The molecule adopts a stable helix formation in solution with a flexible tail between amino acid 140 to 145.
Subject(s)
Adenosine Triphosphate/chemistry , Bacterial Proton-Translocating ATPases/chemistry , Bacterial Proton-Translocating ATPases/ultrastructure , Escherichia coli/enzymology , Models, Chemical , Models, Molecular , Binding Sites , Computer Simulation , Enzyme Activation , Enzyme Stability , Magnetic Resonance Spectroscopy , Protein Binding , Protein Conformation , Protein Subunits/chemistryABSTRACT
The Grb2-related adaptor protein GADS plays a central role during the initial phases of signal transduction in T lymphocytes. GADS possesses N- and C-terminal Src homology 3 (SH3) domains flanking a central Src homology 2 (SH2) domain and a 126-residue region rich in glutamine and proline residues, presumed to be largely unstructured. The SH2 domain of GADS binds the adaptor protein LAT; the C-terminal SH3 domain pairs GADS to the adaptor protein SLP-76, whereas the function of the central region is unknown. High-resolution three-dimensional models are available for the isolated SH2 and C-terminal SH3 domains in complex with their respective binding partners, LAT and SLP-76. However, in part because of its intrinsic instability, there is no structural information for the entire GADS molecule. Here, we report the low-resolution structure of full-length GADS in solution using small-angle x-ray scattering (SAXS). Based on the SAXS data, complemented by gel filtration experiments, we show that full-length GADS is monomeric in solution and that its overall structural parameters are smaller than those expected for a protein with a long unstructured region. Ab initio and rigid body modeling of the SAXS data reveal that full-length GADS is a relatively compact molecule and that the potentially unstructured region retains a significant degree of structural order. The biological function of GADS is discussed based on its overall structure.
