21 / 89
17
Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling Tuesday Speaker Abstracts
Interplay between Weak Nonspecific Electrostatics and Cation-π Interactions Governs the
Peripheral Membrane Binding of a Bacterial Phospholipase
Hanif M. Khan
1
, Cedric Grauffel
1
, Edvin Fuglebakk
1
, Boqian Yang
2
, Tao He
3
, Anne
Gershenson
2
, Mary F. Roberts
3
,
Nathalie Reuter
1
.
1
University of Bergen, Bergen, Norway,
2
University of Massachusetts, Amherst, MA,
USA,
3
Boston College, Chestnut Hill, MA, USA.
Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is an
amphitropic enzyme cleaving GPI-anchored proteins off the outer surface of eukaryotic plasma
membranes. Amphitropic proteins bind specifically and transiently to the surface of cell
membranes, and their functions are regulated upon binding.
It is commonly acknowledged that non-specific electrostatic forces are responsible for their long-
range interactions with membranes. Using continuum electrostatics calculations we show how,
despite having an overall negative charge (-7e), the charge distribution of BtPI-PLC leads to
favorable though quite low electrostatic binding free energy with anionic membranes. The in
silico mutation of a single, key basic residue to alanine diminishes this long-range electrostatic
contribution explaining the significant decrease in the experimentally measured Kd. Multiple 500
ns-long all-atom molecular dynamics simulations of BtPI-PLC docked to mixed bilayers with
varying ratio of zwitterionic lipids show that, once close to the membrane surface, short range
non-specific hydrophobic interactions and specific cation-pi interactions with the N(Me)3 groups
of phosphatidylcholine (PC) lipids of the membrane come into play for BtPI-PLC binding to the
membrane surface[1].
Comparing our simulation results with fluorescence correlation spectroscopy measurements of
the membrane affinity of the wild-type enzymes and of various mutants, we conclude that the
interplay between long-range electrostatics and short range, PC specific cation-pi interactions
governs the specificity of BtPI-PLC for PC-rich membranes. Finally, we suggest that overlooked
cation-pi interactions between membranes and aromatic amino acids of amphitropic proteins
may play an important role not only in membrane binding but also in lipid specificity.
[1] Cation-pi interactions as lipid-specific anchors for phosphatidylinositol-specific
phospholipase-C. C.Grauffel, B.Yang,
T.He, M.F. Roberts, A.Gershenson, N.Reuter*, Journal of
the American Chemical Society (2013) 135(15):5740-50