Figure 5
Involvement of the protein C-end in the network of interactions supporting residue His140 in human GLTP. (a) The His140 side chain makes a key hydrogen bond to the ceramide amide group and multiple contacts with the environment. Ligand and amino acids are shown in stick representation. C atoms are coloured magenta in the lipid, silver in the amino-acid residues surrounding His140 and orange in the partner monomer. Colour codes for O and N atoms are red and blue, respectively. Dashed lines indicate hydrogen bonds, while zigzags are close van der Waals contacts. The C-terminal residue Val209 simultaneously contacts His140 and Pro40* from the partner. (b) The main chain of residue His140 supported by the network of hydrogen bonds involving the C-end of the protein main chain. Designations are as in (a). The circled part of the network highlights the C-end contribution. (c) Disposition of the circled part of the network in proximity to the recognition centre of hsGLTP (the Val209 side chain is skipped). Colour codes are as in (a). (d, e) Transfer activity assays for wtGLTP and mutants. (d) Transfer of the AV-glycolipid by wt-GLTP (blue), Y207L (magenta), [Delta]207 (green) or D48V/[Delta]C-end (orange) as a function of time. The increase in fluorescence emission at 415 nm (AV emission) occurs because of decreased Förster resonance energy transfer when AV-glycolipid is removed from donor vesicles containing 3-perylenoyl PC and is transported to POPC acceptor vesicles (see §[link]2 for details). The AV signal change does not occur in the absence of POPC acceptor vesicles. (e) Transfer activity of GLTP by D48V, Y207L mutations or Y207/C-end deletions. The initial rates of AV-glycolipid departure from the donor vesicles are shown. (f) Expanded view of interactions shown in (b) for the open dimeric arrangement of wtGLTP complexed with monoSF (magenta) versus the locked dimer of the D48V mutant complexed with diSF (cyan). Parts of the partner protein monomer contacting the C-end are highlighted by additional sphere representations in an appropriate colour.  [article HTML]