Probing the oligomeric state and interaction surfaces of Fukutin-I in dilauroylphosphatidylcholine bilayers

Abstract

Fukutin-I is localised to the endoplasmic reticulum or Golgi apparatus within the cell, where it is believed to function as a glycosyltransferase. Its localisation within the cell is thought to to be mediated by the interaction of its N-terminal transmembrane domain with the lipid bilayers surrounding these compartments, each of which possesses a distinctive lipid composition. However, it remains unclear at the molecular level how the interaction between the transmembrane domains of this protein and the surrounding lipid bilayer drives its retention within these compartments. In this work, we employed chemical cross-linking and fluorescence resonance energy transfer measurements in conjunction with multiscale molecular dynamics simulations to determine the oligomeric state of the protein within dilauroylphosphatidylcholine bilayers to identify interactions between the transmembrane domains and to ascertain any role these interactions may play in protein localisation. Our studies reveal that the N-terminal transmembrane domain of Fukutin-I exists as dimer within dilauroylphosphatidylcholine bilayers and that this interaction is driven by interactions between a characteristic TXXSS motif. Furthermore residues close to the N-terminus that have previously been shown to play a key role in the clustering of lipids are shown to also play a major role in anchoring the protein in the membrane.

Fig. 1

Analysis of chemical cross-linking of reconstituted FK1TMD peptide using SDS-polyacrylamide gel electrophoresis (PAGE). FK1TMD was reconstituted into DLPC vesicles at lipid-to-peptide molar ratio of 100:1 and cross-linked using DSS. Cross-linked and uncrossed samples (containing ~7 μg FK1TMD) were run on 16% tricine SDS-PAGE gel and visualised after staining with SYPRO Orange. Cross-linked sample was visualised as two distinct bands: monomer (unlinked) and dimer. Similar results were obtained using 0.05% glutaraldehyde as cross-linker

Fig. 2

Fluorescence emission spectra of FK1TMD in DLPC bilayers with constant lipid-to-peptide ratio of 100:1 (solid lines). The ratio of FITC-labelled FK1TMD to RBITC-labelled and unlabelled FK1TMD was maintained at 1:3, whilst the ratio of RBITC-labelled FK1TMD and unlabelled peptide was varied from 0% to 100% in steps of 25%. Rhodamine emission spectra follow excitation at 492 nm showing no emission at 520 nm (solid line)

Fig. 3

Observed quenching of emission spectrum from FITC-labelled FK1TMD at 520 nm in response to increase concentrations of the FRET acceptor RBITC-labelled FK1TMD within the DLPC bilayers (lipid-to-peptide ration, 100:1). The ratio of FITC-labelled FK1TMD to RBITC-labelled and unlabelled FK1TMD was maintained at 1:3, whilst the ratio of RBITC-labelled FK1TMD and unlabelled peptide was varied from 0% to 100%. Data normalised to give F/F ₀ of 1.0 in the absence of any RBITC-labelled FK1TMD acceptor. Line indicates best fit (r ² = 0.98) to the linear quenching dependency characteristic of dimer formation

Fig. 4

CG simulations of dimer formation. The panel on the left shows the inter-helical distance as a function of time during the five CG simulations. In each case the dimer is stable after 0.5 μs. A representative snapshot of the individual monomers at the start of the simulation and a dimer are shown in backbone format (purple). The image on the right shows the dimer embedded within a DLPC lipid bilayer (lipids are green). Some residues with high propensity to interact with lipids are shown in space-filling format (R3 and K6 are cyan, K29 and R35 are orange)

Fig. 5

Inter-helix interactions of residues S17 and S18. The plot on the left shows the distance between these residues as a function of time in the three atomistic simulations. A representative snapshot of the dimer backbone (purple) is shown on the right. Residues S17 and S18 are shown in space-filling format (carbon atoms are cyan, nitrogen atoms are blue, oxygen atoms are red and polar hydrogen atoms are white). The same residues are shown close-up in stick representation in the bottom right panel, with hydrogen bonds shown in green

Fig. 6

Examples of lipid–protein interactions. The panel on the left shows an electrostatic interaction between the guanidinium group R3 of a Fukutin monomer and a phosphate group of a DLPC lipid molecule. The panel on the right shows a hydrogen-bond interaction between N5 of a Fukutin monomer and a phosphate group of a DLPC lipid