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 kcal͞mol at 25°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.

The Role of Intermediary Domain of MyD88 in Cell Activation and Therapeutic Inhibition of TLRs

“…The anomalously elongated C-terminal helix of the MyD88 DD may be a result of the crystallization conditions. Interestingly, it has been reported that nonpolar cosolvent used for crystallization induced formation of an extended helix of DD of Pelle that is absent in the solution (38). In support of this proposal, the crystal structure of the complex between DDs of the Drosophila-specific adaptor protein Tube and Pelle, an IRAK4 orthologous kinase (Fig.…”

Membrane-induced Lever Arm Expansion Allows Myosin VI to Walk with Large and Variable Step Sizes

“…The overall conformation of Bcl-2 can be transformed from a compact eight-helical bundle in aqueous solution into a transmembrane ion channel structure upon binding to lipid membranes (56,57). Death domains have been observed to undergo membrane-induced helix opening (58), and this conformational change might be responsible in part for the further oligomerization of death domains and the subsequent activation of the Fas-FADD death-inducing signaling complex pathway (59). In lipid membrane environments, the ALPS (ArfGAP1 lipid packing sensor) motif in a number of proteins can even transform their conformations from totally unstructured states to ␣-helical structures, allowing the domains to sense membrane curvatures (60,61).…”

Solution structure of the isolated Pelle death domain

“…To ensure that the changes seen in the HSQC spectrum of Pelle‐DD when in complex with Tube‐DD could be interpreted as arising from complex formation and not necessarily because of differences between the structures of Pelle in both states a high resolution structure of the isolated Pelle‐DD was required. Previous attempts at obtaining such data by X‐ray crystallography were unsuccessful [43] and consequently, NMR spectroscopy was used.…”