title: Cosolvent-induced transformation of a death domain tertiary structure.
abstract: The death domain (DD) of the protein kinase Pelle adopts a six-helix bundle fold in the crystal structure of the complex with its dimerization partner, Tube-DD. However, in crystals obtained from a solution of 45% 2-methyl-2,4-pentanediol (MPD), the C-terminal half of Pelle-DD folds into a single helix, and the N-terminal half of the molecule is disordered. The helical segment forms an antiparallel dimer with the corresponding helix of a symmetry-related molecule, and together they form extensive lattice interactions similar in number, composition, and buried surface to those in the six-helix bundle of the native fold. Secondary structure analysis by heteronuclear nuclear magnetic resonance spectroscopy (NMR) demonstrates that Pelle-DD adopts a six-helix bundle fold in aqueous solution. The fold is perturbed by MPD, with the largest chemical shift changes in one helix and two loop regions that encompass the Tube-DD binding site. Pelle-DD is stable to urea denaturation with a folding free energy of 7.9 kcal/mol at 25 degrees C but is destabilized, with loss of urea binding sites, in the presence of MPD. The data are consistent with a cosolvent denaturation model in which MPD denatures the N terminus of Pelle-DD but induces the C terminus to form a more compact structure and aggregate. A similar perturbation in vivo might occur at the plasma membrane and could have consequences for Pelle-mediated regulation. Generally, crystallographers should be aware that high concentrations of MPD or related cosolvents can alter the tertiary structure of susceptible proteins.
authors.affiliation: The Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9050, USA.
authors.forename: Tsan
authors.initials: T
authors.lastname: Xiao
authors.forename: Kevin H
authors.initials: KH
authors.lastname: Gardner
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Sprang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents; D014508:Urea
chemical_list: D029721:Drosophila Proteins; D011994:Recombinant Proteins; D012997:Solvents; D014508:Urea; C079896:pll protein, Drosophila; D017346:Protein Serine-Threonine Kinases