Structural characterization of the C2 domain of novel protein kinase Cε

Abstract

Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKC:-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca 21 -independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H 2 O and D 2 O buffers, b sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D 2 O showed a broadening in the amide I H band starting at 45 8C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm 21 which was assigned to aggregation and a second one at 1645 cm 21 , which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKC:-C2 domain has preferencial affinity for PIP 2 although it exhibits maximal binding activity for phosphatidic acid when 100 mol% of this negatively charged phospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a T m of 49 8C and a calorimetric denaturation enthalpy of 144.318 kJ´mol 21 . However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.

Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

“…For example, it is well established that PLD activation induces an increase of phosphatidic acid in biological membranes [27] and this could be a way of activating PKCε. As it was demonstrated previously [23] the PKCε-C2 domain has an important affinity for phosphatidic acid vesicles although the secondary structure of the domain did not change upon lipid binding, in contrast with PKCα-C2 domain that underwent significant changes ( [23] and this work). These results correlate well with the need for the PKCα to penetrate the membrane to be fully activated, the result of which could be the structural reorganization seen by IR.…”

“…The spectrum was obtained and curve fitting was carried out for the PKCε-C2 domain bound to vesicles containing 100 mol% of phosphatidic acid in D 2 O at 25 • C in the 1780- 1600 cm −1 region. The curve fitting data point to a secondary structure very similar to that obtained in the absence of lipids [23] which suggests that no differences or changes in the secondary structure occur when lipid binding takes place. It is interesting to note that these results are different from those observed for the PKCα-C2 domain, which showed an increase in the component representing unordered and open loop structures upon lipid binding [24].…”

“…A further significant change (Fig. 7C) concerned the component at 1686 cm −1 , which shifted to 1681 cm −1 at the same time that the percentage of its area increased from 4% at 25 • C to 10% at 75 • C [23]. These data suggest that there is only a small reorganization in the initial conformation of the β-turns, for example, the component at 1651 cm −1 slightly shifted to 1653 cm −1 at 40 • C. Furthermore, the percentage of the area decreased from 21% at 25 • C to 14% at 40 • C and stayed constant during denaturation, indicating that this component, which is attributed to α-helix or large loops, is very stable during the heating process.…”

“…It is interesting to note that in spite of sharing a core β-sheet with many other C2 domains, including the PKCα-C2 domain, IR detected significant differences between this last domain and the PKCε-C2 [23,24]. For example, the component at 1644 cm −1 assigned to open loops and unordered structure in the PKCα-C2 cannot be found in the PKCε-C2 domain.…”

“…Nishizuka and Ono (Kobe University, Kobe, Japan). The resulting PCR fragment were subcloned using sites of the bacterial expression vectors, pET28, in which the inserts were fused to 6His tag, respectively as described previously [23,24]. All constructs were confirmed by DNA sequencing.…”