Helix orientations in membrane-associated Bcl-XL determined by 15N-solid-state NMR spectroscopy

Abstract

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x(L) is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x(L) associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x(L) its membrane interactions are weak. In the presence of membranes Bcl-x(L) can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x(L) hydrophobic binding pocket becomes accessible for protein-protein interactions whilst the structure of the N-terminal region remains intact.

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy

“…4C) where the hydrophobic peptide-peptide interactions are replaced by peptide-membrane interactions within the bilayer interface. Related conformational changes have been demonstrated for the pore-forming domains of colicins when interacting with phospholipid membranes (28) and for domains of proteins of the Bcl-2 family (29).…”

Investigations of the synergistic enhancement of antimicrobial activity in mixtures of magainin 2 and PGLa

“…The deuterium solid-state NMR spectra of the 2 H3-Ala10-site of PGLa exhibit a quadrupolar splitting of about 12 kHz in the presence 31 P solid-state NMR spectra of the phospholipids show that, despite the well-oriented 15 N spectra of the same sample, considerable orientational dispersion of the phospholipid head groups with a chemical shift anisotropy of about 37 ppm (Figure 5C, F). Although the main intensity corresponds to phospholipids that align with their long axis parallel to the sample normal (24 ppm) major intensities up to -13 ppm suggest an additional topological distribution as one would observe from spherical (powder pattern) or cylindrical structures[54,55]. These additional intensities are already obvious in supported lipid bilayers made from E. coli lipid extracts(Fig.…”

A dynamic view of peptides and proteins in membranes

“…When the structure and interactions of the antiapoptotic Bcl-x L were investigated in the presence of membranes by solid-state NMR spectroscopy the data agreed with a type I membrane protein where the global fold encompassing helices 1 -8 remains intact (Fig. 1B) and where the most C-terminal helix anchors the protein in the membrane [40]. Membrane insertion is thus correlated with conformational changes which upon interaction of the C-terminal helix with the membrane liberates a hydrophobic binding pocket for interactions with other members of the Bcl-2 family.…”

“…In this configuration the hydrophobic region is localized about 10 above the bilayer center in agreement with the amphipathic distribution of polar-charged and hydrophobic residues. The peptides thereby 3032 B. Bechinger Structure and dynamics of membranes reflect the interactions and topologies that have also been suggested for individual helices of the Cterminal domains of several colicins or of members of the Bcl-2 family of proteins [40,63,67,108]. In addition to the equilibria that govern the polypeptide interactions within the membrane (in-plane $ transmembrane $ TM oligomers), monomeric and aggregated states also interchange within the water phase (Fig.…”

“…Although variable alignments have also been observed when investigating membrane-associated peptides [40,76], this concept is less common for larger membrane proteins. However, it remains possible that such loosely folded membrane structures occur more often but escape our attention as such conformational flexibility is difficult to visualize by crystallographic techniques.…”