Configuration of PKCα-C2 Domain Bound to Mixed SOPC/SOPS Lipid Monolayers

Abstract

X-ray reflectivity measurements are used to determine the configuration of the C2 domain of protein kinase Calpha (PKCalpha-C2) bound to a lipid monolayer of a 7:3 mixture of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of PKCalpha-C2 and a slab model representation of the lipid layer. The configuration of lipid-bound PKCalpha-C2 is described by two angles that define its orientation, theta = 35 degrees +/- 10 degrees and phi =210 degrees +/- 30 degrees, and a penetration depth (=7.5 +/- 2 A) into the lipid layer. In this structure, the beta-sheets of PKCalpha-C2 are nearly perpendicular to the lipid layer and the domain penetrates into the headgroup region of the lipid layer, but not into the tailgroup region. This configuration of PKCalpha-C2 determined by our x-ray reflectivity is consistent with many previous findings, particularly mutational studies, and also provides what we believe is new molecular insight into the mechanism of PKCalpha enzyme activation. Our analysis method, which allows us to test all possible protein orientations, shows that our data cannot be explained by a protein that is orientated parallel to the membrane, as suggested by earlier work.

Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4

“…The lipid-only data yielded mean values of 13.4 ± 0.1 Å and 10.5 ± 0.4 Å for the thickness of the lipid tail and head regions, respectively; the electron density values were 0.231 ± 0.004 electrons/Å 3 for the tail region and 0.453 ± 0.004 electrons/Å 3 for the head group region. These values are in general agreement with previously measured values of similar systems, allowing for small variations resulting from different buffer conditions (32,33). Table S1 provides the specific parameter values associated with the best fits to the lipid-only and +1 μM Tim4 data.…”

“…This intensity versus incident angle relationship is essentially an interference pattern produced by variations in the 1D electron density profile along the dimension perpendicular to the reflecting interface, averaged over the in-plane dimension parallel to the interface (31). Given that we know the general molecular structure of both the lipid monolayer and the protein (from the crystal structure), this 1D electron density profile is, in principle, sufficient to determine a precise orientation of a protein bound to a membrane, presuming the electron density of the protein is not symmetrically distributed (32,33).…”

“…The lipids were deposited to an area/molecule roughly equivalent to that of a SOPC bilayer (∼65 Å 2 /molecule) (34). The SO lipids were used in keeping with previous studies to help minimize oxidative damage of the unsaturated tail by X-ray radiation (32,33). The reflected intensity as a function of momentum transfer vector [Q z = 4π sin (α)/λ, where α is the incident angle and λ is the X-ray wavelength] for lipid monolayers with or without 1 μM Tim4 is shown normalized by Fresnel reflectivity from an ideal air/buffer interface ( Fig.…”

“…2B was generated using recently developed methods for incorporating protein crystal structures to fit reflectivity data (32,33). Under this approach, a reduced χ 2 map depicting the fit quality as a function of protein orientation was generated.…”

“…To determine the precise orientation of Tim4 bound to a lipid monolayer, we used a recently developed extension of the two-box model that uses existing protein crystal structure information (32,33). The analysis performed here follows closely that of these previous studies and uses essentially the same custom C code for calculating electron density profile and performing iterative fitting (done in the Cplot program, available at www.certif.com/ content/cplot/).…”