Structural basis of membrane bending by the N-BAR protein endophilin

Abstract

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.

Figure 1. Endophilin Scaffold Structure (see also figure S1)

(A) classaverages (top) and reconstructed volumes (bottom) of membrane tubules decorated with full-length endophilin. The size of the scale bar is the same in corresponding reconstructions and classaverages. The bilayer is colored in yellow, protein is shown in green. Apparent holes seen in the bilayer of the 32nm tube reconstruction are due to the thresholding level, which was chosen to emphasize the molecular envelope of the protein component. (B) model of endophilin lattice; top view (left), and cross-section perpendicular to tube axis (right). The high-resolution crystal structure of rat endophilin A1 (pdb: 1ZWW, orange) was fitted into the reconstructions of full-length endophilin tubule. Only one full dimer of the BAR-domain core is shown for clarity. The disposition of H0 (magenta) and insert (cyan) helices is indicated by cylinders. The molecular envelope (grey mesh) suggested that H0-helices from adjacent BAR-domain dimers pair in an antiparallel fashion. At the same contouring threshold, one of the insert helices is not fully accounted for, reflecting the partially disordered state of this element in our reconstructions. The length of the vertical thin blue lines (right panel) is 30Å and marks the approximate positioning of the hydrophobic core of the bilayer. At the chosen contouring threshold we did not observe density for the leaflet that is in direct contact with the BAR-domain. This indicates that the bilayer is highly disordered and stressed.

Figure 2. The N-Terminal helix H0 plays a crucial role in lattice formation (see also figure S2)

(A) Starting (top) and final (bottom) snapshots of coarse-grained molecular dynamics simulations of endophilin oligomers (N-BAR on the left and H0 deleted BAR on the right). Green represents the coiled-coil BAR scaffold, blue represents the H0 helix and white represents the insert helix (see Figure S4). (B) Free energy profile for anti-parallel H0 pairs as function of displacement against each other. The helix pair is shoulder-to-shoulder when the offset is 0Å. (C) Two stable configurations for the helix pair. The label “a” (at -13Å) indicates the configuration sampled in the CG simulations. The free energy was calculated from the umbrella sampling data by weighted histogram analysis method and error bar was calculated by the bootstrap method. The orange line is for reference purpose only, showing that the energy is slightly higher in the direction of positive displacement. (D) cryoEM images (top) and single particle classaverages (bottom) of endophilin wildtype (wt) and partial H0-deletion mutants of endophilin (Δ2-10 and Δ2-13). For the Δ2-10 mutant, 294 overlapping segments contributed to the average shown, out of 1641 segments in the data set. For the Δ2-13 mutant, 156 overlapping segments contributed to the shown average, out of 561 in the data set. The lower numbers compared to wild type endophilin averages were due to the much lower number of well preserved tubes that were observed for these mutant endophilins. The scale bar in all top cryoEM images is 25nm. The scale bar in all single particle classaverages is 5nm. Tubules of endophilin (wt) exhibit a characteristic ‘studded’ appearance indicating a coherent protein coat with a repeat distance of ~5nm. This spacing is not detectable in tubules formed by both H0 truncation mutants of endophilin (Δ2-10 and Δ2-13), suggesting a loss of coherence within the protein coat.

Figure 3. Interactions between H0-helices are non-specific and degenerate (see also figure S3)

(A) Crosslinking efficiency for 12 unique Cys substitutions along H0. All data are presented as mean + S.E.M (triplicates), (B) Impact of synthetic H0 peptides on crosslinking of unique Cys-substituted endophilin mutants. Striped bar refers to crosslinking in absence of peptide; grey bar: 22-residue wild-type endophilin H0 peptide; black bar: phosphomimetic mutant peptide (T14D). All but one mutant (S2C) showed a significant reduction in the crosslinking efficiency in the presence of either H0 peptide. In these experiments, peptides were used at 2mM concentrations. Significance levels: *p>0.05, **p>0.01. tested with Student's t-test (C) negative stain EM images of tubulation reactions with liposomes in the absence (left panels) or presence (right panels) of 2mM of endophilin H0-peptide (residues 1-22). Concentrations of 2mM effectively inhibited the ability of endophilin to bend membranes. This block of tubule formation was observed with amphiphysin 2 as well, an N-BAR protein similar to endophilin. In contrast, tubulation by the F-BAR protein FBP17 was less affected, indicating that the inhibition of N-BAR dependent curvature generation was caused by a disruption of H0:H0 interactions between N-BAR dimers. In all images, scale bar=2μm.

Figure 4. Disposition of SH3-domains

(A) Partial overlay of the envelops for reconstructions of full length endophilin (black) and the endophilin N-BAR domain only (red). Shown are the overlays for tubes of 28nm (left) and 25nm (right). An additional volume element was observed in the full-length endophilin reconstruction of the 28nm tube. (B) The volume of the additional density was large enough to accommodate the high-resolution crystal structure of the rat endophilin A1 SH3-domain dimer (pdb: 3IQL), which was fitted without any modifications to the coordinates. (C) The positions of Cys294 (pink) and Thr320 (yellow) are marked in the structure (left). The black scale bar above the dimer is 10Å. Shown to the right are coommassie stained, non-reducing SDS-PAGE gels of wild-type endophilin, and a Thr320Cys mutant after tubulation and cross-linking; M: molecular weight marker. In the crystal structure of the SH3-domain dimer, the pair of Cys294 residues is just over 10Å apart, slightly too far to efficiently crosslink. However, substituting Thr320 for Cys should and did allow efficient crosslinking of endophilin based on the appearance of the ~75kD endophilin dimer. This is consistent with the idea that in some cases, SH3-domains dimerize above the BAR-domain scaffold.

Figure 5. N-BAR Scaffolds Compartimentalize the Membrane Surface For Interactions with Downstream Effectors

In contrast to F-BAR lattices, endophilin lattices expose large membrane surface areas. Shown here is how the size of these areas is matched to the size of membrane binding domains from downstream effectors that are recruited by endophilin. Specifically, the PH-domain of dynamin (pdb: 2DYN) and the inositol polyphosphate phosphatase catalytic domain (IPP5C) of synaptojanin from S. pombe (pdb: 1I9Z) are perfectly accommodated within the constraints of the endophilin lattice.