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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes
A Transmembrane Helix Induces Membrane Fusion Through Lipid Binding and Splay Holger A. Scheidt, Katja Kolocaj, Julie Veje Kristensen, Daniel Huster, and Dieter Langosch J. Phys. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018
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The Journal of Physical Chemistry Letters
1 jz-2018-00859x_revised manuscript
A Transmembrane Helix Induces Membrane Fusion through Lipid Binding and Splay Holger A. Scheidt Dieter Langosch [a]
[a]#
, Katja Kolocaj
[b]#
, Julie Veje Kristensen
[b]
Daniel Huster
[a]*
, and
[b]*
Institute for Medical Physics and Biophysics, Leipzig University, Härtelstr. 16-18, 04107
Leipzig, Germany E-mail:
[email protected] [b]
Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner
Berg 3, 85354 Freising and Munich Center For Integrated Protein Science (CIPSM), Germany E-mail:
[email protected] #
Both authors contributed equally
*
Correspondence author
Dieter Langosch, Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising and Munich Center For Integrated Protein Science (CIPSM), Germany, E-mail:
[email protected], Ph: +49-8161-713500
This work was supported by the Deutsche Forschungsgemeinschaft (LA699/16-1 to DL and HU 720/15-1 to DH) and the Center of Integrative Protein Science Munich (CIPSM).
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2 Abstract
The fusion of biological membranes may require splayed lipids whose tails transiently visit the headgroup region of the bilayer, a scenario suggested by molecular dynamics simulations. Here, we examined the lipid splay hypothesis experimentally by relating liposome fusion and lipid splay induced by model transmembrane domains (TMDs). Our results reveal that a conformationally flexible transmembrane helix promotes outer leaflet mixing and lipid splay more strongly than a conformationally rigid one. The lipid dependence of basal as well as of TMD-driven lipid mixing and splay suggests that the cone-shaped phosphatidylethanolamine stimulates basal fusion via enhancing lipid splay and that the negatively charged phosphatidylserine inhibits fusion via electrostatic repulsion. Phosphatidylserine also strongly differentiates basal and helix-driven fusion which is related to its preferred interaction with the conformationally more flexible transmembrane helix. Thus, the contribution of a transmembrane helix to membrane fusion appears to depend on lipid binding which results in lipid splay.
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The Journal of Physical Chemistry Letters
3 Membrane fusion supports many cellular processes and is mediated by dedicated membrane proteins.1-2 Mechanistically, fusion starts with the formation of an hourglass-shaped stalk structure; this is followed by outer leaflet (OL) mixing and completed by fusion pore formation and inner leaflet (IL) mixing.3-5 It is currently unclear at which stage and to which extent the transmembrane domains (TMDs) of the fusogenic single-span proteins contribute to the mechanism of fusion.6-9 Previously, the function of fusogenic proteins has been linked to the conformational flexibility of their TMD helices based on an overrepresentation of helixdestabilizing amino acids, such as Gly or the β-branched residues Ile and Val within the TMDs.10 This link is supported by mutational studies of various full-length fusogenic protein TMDs in cellulo 6 and by the finding that synthetic peptides representing such TMDs proved to be fusogenic in vitro, depending on their helix backbone flexibility.11-14 In line with these observations, systematically increasing the Val/Leu ratio of LV-TMDs, a designed TMD model system that supports fusion in vitro 15 and in cellulo 16, increases TMD helix flexibility as well as fusogenicity.15-19 The mechanism of how conformational flexibility enhances fusogenicity has remained elusive, however.
It is also widely recognized that membrane fusion is modulated by different types of lipids. Depending on their shape, their internal flexibility, and their charge, lipids can affect the curvature, hydration, and/or fluidity of a membrane
20
Also, lipids have been proposed to
bind, cluster, and recruit fusogenic proteins to membrane microdomains
21-22
Another
important function of lipids has been implied by a number of molecular dynamics simulations that suggested a ‘pre-stalk intermediate’ in the pathway leading to fusion. In this model, lipids transiently splay such that their tails are exposed to the bilayer surface, ready to contact another bilayer. This has been modelled in purely lipidic environments,23-29 or in the vicinity of membrane-fusogenic soluble peptides30-31 or TMDs.32-33 That lipid tails are highly dynamic and frequently visit the headgroup region of protein-free membranes has also been shown experimentally.34-36
Here, we relate spontaneous as well as TMD-induced liposome fusion to the respective levels of lipid binding and lipid splay in different lipid environments. Lipid mixing as a readout for fusion was studied using a standard fluorescence dequenching assay after reconstituting the low-fusogenicity L16 (K3WL16K3) or the highly fusogenic LV16 (K3W[LV]8K3) TMD into liposomes (median size = 124 ± 6.1 nm, Figure S1). Lipid binding was monitored by Trp
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4 quenching by brominated lipids. Lipid splay was investigated by high resolution solid state 1H NOESY NMR spectroscopy under magic-angle spinning (MAS) conditions.
First, lipid mixing was assessed using a standard fluorescence dequenching assay which is based on decreasing fluorescence resonance energy transfer between 1.5 mol%, each, of the membrane-bound
fluorophores
yl)hexadecylphosphatidylethanolamine
(NBD-PE)
N-(7-nitro-2,1,3-benzoxadiazol-4and
N-(lissamine
rhodamin
B
sulfonyl)hexadecylphosphatidylethanolamine (Rh-PE) upon fusion of fluorescence-labeled donor liposomes with unlabeled acceptor liposomes containing the TMDs. Lipid mixing was compared in different mixtures of dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylserine (DOPS), and dioleoyl phosphatidylethanolamine (DOPE). The exemplary ‘total’ fusion kinetics (that comprise OL and IL mixing), as shown for DOPC/DOPS/DOPE (3:1:1) liposomes (Figure 1 A), confirm the differential fusogenicities of LV-TMDs (blank < L16
