PDBsum entry 2c08

Transferase

PDB id: 2c08

Contents

Protein chain

204 a.a. *

* Residue conservation analysis

PDB id:

2c08

Name:

Transferase

Title:

Rat endophilin a1 bar domain

Structure:

Sh3-containing grb2-like protein 2. Chain: a. Fragment: bar domain, residues 1-143. Synonym: endophilin a1, sh3 domain protein 2a, sh3p4. Engineered: yes

Source:

Rattus norvegicus. Rat. Organism_taxid: 10116. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.90Å R-factor: 0.280 R-free: 0.295

Authors:

J.L.Gallop,H.M.Kent,H.T.Mcmahon,P.R.Evans

Key ref:

J.L.Gallop et al. (2006). Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J, 25, 2898-2910. PubMed id: 16763559 DOI: 10.1038/sj.emboj.7601174

Date:

26-Aug-05 Release date: 14-Jun-06

Protein chain

O35179  (SH3G2_RAT) -  Endophilin-A1 from Rattus norvegicus

Seq: 352 a.a.

Struc: 204 a.a.

Enzyme reactions

• Enzyme class: E.C.2.3.1.- - ?????

DOI no: 10.1038/sj.emboj.7601174

EMBO J 25:2898-2910 (2006)

PubMed id: 16763559

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

J.L.Gallop, C.C.Jao, H.M.Kent, P.J.Butler, P.R.Evans, R.Langen, H.T.McMahon.

ABSTRACT

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.

Selected figure(s)

Figure 2.
Figure 2 Membrane insertion and orientation of endophilin N-terminal amphipathic helix. (A) Oxygen (red circles) and NiEDDA (green squares) accessibilities ( ) of membrane-bound N-BAR domain as a function of label position. The graph below shows a ln( ratio) plot ( ) showing the differential access of colliders to the spin label and the penetration of hydrophobic residues into the membrane. The periodic oscillation is indicative of a helical structure. Equivalent maxima indicate that the helix lies planar to the membrane. (B) Helical wheel representation showing hydrophobic and charged faces. (C) Model of the amphipathic helix, residues 1–16 with hydrophobic residues coloured green and surface charge potential also shown. (D) Model of the N-BAR amphipathic helix to scale with PtdIns(4,5)P[2] and PtdSer lipids showing the depth of penetration of the helix as calculated from data in (A) and penetration measurements, described in Materials and methods.

Figure 7.
Figure 7 High membrane curvature promotes membrane fusion. (A) Example of endophilin N-BAR tubules made from liposomes extruded to a size cutoff of 400 nm. (B) Emission spectra from mixed liposomes in the absence and presence of calcium (which promotes fusion) and Triton (to obtain total donor fluorescence). See Materials and methods for details of the assay. (C) FRET assay of membrane fusion, showing dilution of the FRET pair into unlabelled liposome in the presence of the N-BAR (see Results). The N-BAR control has no unlabelled liposomes and thus there can be no dilution of the FRET pair. The BAR domain does not lead to membrane fusion. As highly curved membranes are more fusogenic, we believe that the fusion seen with the N-BAR domain is a readout of the efficiency of curvature generation. (D) Comparison of mutants and WT N-BAR domains at 55 M in the fusion assay. Values displayed in the bar chart s.e.m. are the difference in the ratios of emission maxima between experiment (mixed liposomes (530/585 nm)) and controls (uniformly labelled liposomes (530/585 nm)) (see Results). Studentʼs t-test ^*P<0.001, ^**P<0.2.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2006, 25, 2898-2910) copyright 2006.

Figures were selected by an automated process.