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Proc Natl Acad Sci U S A. Apr 21, 2009; 106(16): 6603–6607.
Published online Apr 3, 2009. doi:  10.1073/pnas.0813099106
PMCID: PMC2672498
Biochemistry

Structural and mechanistic insights into the association of PKCα-C2 domain to PtdIns(4,5)P2

Abstract

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca2+-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCα-C2 domain in complex with Ca2+, 1,2-dihexanoyl-sn-glycero-3-[phospho-l-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P2] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P2 occupies the concave surface of strands β3 and β4. Strikingly, the structure reveals a PtdIns(4,5)P2-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P2 severely impaired the ability of PKCα to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P2 is presented.

Keywords: calcium phosphoinositides, peripheral membrane proteins

The C2 domains are considered peripheral proteins that are water-soluble and associate reversibly with lipid bilayers. Recently, evidence has demonstrated that some of these domains are able to interact with the inositol phospholipid 1,2-diacyl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P2] (14), which is able to directly participate in a myriad of functions, including cell signaling at the plasma membrane, regulation of membrane traffic and transport, cytoskeleton dynamics, and nuclear events (5, 6). Despite the number of C2 domain 3D structures currently available, questions about how they interact with the different target phospholipids, their precise spatial position in the lipid bilayer, and their role in transmitting signals downstream have yet to be explored.

The main role of the C2 domain in classical PKCs (cPKCs) is to act as the Ca2+-activated membrane-targeting motif (7, 8). The 3D structure of these C2 domains comprises 8 antiparallel β-strands assembled in a β-sandwich architecture, with flexible loops on top and at the bottom (912). This C2 domain displays 2 functional regions: the Ca2+-binding region and the polybasic cluster. The former is located in the flexible top loops, binds 2 or 3 Ca2+ ions, depending on the isoenzyme (10, 11, 13, 14), and interacts with 1,2-diacyl-sn-glycero-3-[phospho-l-serine] (PtdSer) (11, 15, 16). The second region is a polybasic cluster that is located at the concave surface of the C2 domain formed by strands β3 and β4. Recent studies indicate that this region might bind specifically to PtdIns(4,5)P2 in a Ca2+-dependent manner (1, 1721).

To gain insight into the structural and functional basis for the PtdIns(4,5)P2-dependent membrane targeting of the PKCα-C2 domain, we determined the 3D structures of the ternary and quaternary complexes of the C2 domain of PKCα, crystallized in presence of Ca2+ and PtdIns(4,5)P2 or Ca2+, PtdIns(4,5)P2 and PtdSer. In addition, the crystallographic results were validated in living cells by site-directed mutagenesis of the residues involved in the PtdIns(4,5)P2-C2 domain interaction. Noteworthy, most of the residues found in the phosphoinositide interaction are highly conserved among C2 domains of topology I, and a general mechanism for membrane docking of C2 domains regulated specifically by PtdIns(4,5)P2 is proposed.

Results and Discussion

PtdIns(4,5)P2 Binds Specifically to the β3–β4 Groove in the PKCα-C2 Domain.

To understand how the C2 domain of cPKCs interacts with its 2 targets: PtdSer and PtdIns(4,5)P2 2 different crystals forms were obtained of the recombinant PKCα-C2 domain. One crystallized in the presence of Ca2+ and PtdIns(4,5)P2, and the other crystallized in the presence of Ca2+, PtdIns(4,5)P2, and PtdSer. The final electron density maps confirmed the presence of the phospholipid ligands in both structures (Fig. 1 and Table S1).

Fig. 1.
Structure of PKCα C2 domain bound to PtdIns(4,5)P2. (A) The PKCαC2-Ca2+-PtdIns(4,5)P2 ternary complex. (B) The PKCαC2-Ca2+-PS-PtdIns(4,5)P2 quaternary complex. The C2 molecule is shown in blue. The calcium ions located at the tip ...

The 3 Ca2+ ions found at the calcium-binding pocket in the 2 complexes are located in equivalent positions to the calcium sites Ca1, Ca2, and Ca3, as described (16, 22) and in Fig. S1. In the PKCαC2-Ca2+-PtdIns(4,5)P2 structure, a strong peak of extra electron density in this region was interpreted by the presence of a phosphate ion, completing the coordination of Ca1 (Fig. S1A). In the PKCαC2-Ca2+-PtdSer-PtdIns(4,5)P2 structure, a more elongated density appeared close to Ca1 that was interpreted as corresponding to a partially-ordered phosphoserine head group of PtdSer (Fig. S1B).

Well-defined extra electron densities were found, in both complexes, within the concave surface formed by strands β3 and β4 of the C2 domain, the β3–β4 groove. These densities were clearly explained by the presence of the inositol(1,4,5)-trisphosphate (InsP3) head groups of PtdIns(4,5)P2, occupying the cavity (Fig. 1 A and B). When the InsP3 head group of PtdIns(4,5)P2 is positioned in the region, all 3 phosphate groups and, to a lesser extent, the polyalcohol moiety interact with the C2 domain (Fig. 1). Three of the 4 conserved lysines within the polybasic cluster are involved in interactions with PtdIns(4,5)P2: Lys-197 of β3 forms 2 polar bonds with the phosphate 5 moiety and the hydroxyl group O6 of the inositol ring, Lys-209 of β4 forms a salt bridge with the phosphate 1 that remains partially exposed to the solvent, and Lys-211, also from β4, interacts with the phosphate 4 moiety. In addition, 3 other residues participate in the interaction: the hydroxyl group of Tyr-195 is hydrogen-bonded to phosphates 4 and 5. The side chain of Asn-253 forms a weak hydrogen bond with the phosphate 5 moiety (distance 3.51 Å). Finally, Trp-245, located in the proximity, also contacts phosphate 5 (Fig. 1C). These findings are in accordance with the biochemical results obtained previously, because mutations abolishing the calcium-binding region affected only the PtdSer binding (15, 23, 24), and mutations abolishing the concave region formed by strands β3 and β4 altered only the PtdIns(4,5)P2 interaction (1, 16, 19). In addition, the interaction between aromatic residues and phosphoinositide head groups has also been observed in other peripheral membrane proteins like some pleckstrin homology domains (25).

The results obtained above immediately suggested that an InsP3 molecule would also bind to the C2 domain in a similar way to the PtdIns(4,5)P2 molecule. Because of the importance of InsP3 in the signal transduction pathways involving PKCα activation, we examined the thermodynamics of soluble InsP3 binding to the C2 domain of PKCα by isothermal titration calorimetry at 25 °C in the presence of saturating Ca2+. The binding data demonstrated that there is 1 binding site but that is not completely occupied (n = 0.51 ± 0.14) with a KD of 19.8 ± 1.7 μM (Fig. S2). These results demonstrate that the affinity of the C2 domain for PtdIns(4,5)P2 (KD = 1.8 μM in ref. 19) is higher than for soluble InsP3 or PtdSer (KD = 18 μM in ref. 20), suggesting that other phosphoinositide moieties and/or the orientation adopted by the PtdIns(4,5)P2 molecule in the membrane might contribute to increase this affinity.

Several studies have demonstrated that the unphosphorylated inositol ring is oriented perpendicular to the bilayer surface (26), whereas the double phosphorylation in C4 and C5 of the inositol induces a bend toward the bilayer surface (26, 27). This orientation would be similar to the one found in the crystal structure presented here and thus would be compatible with a perfect docking of the C2 domain in the membrane interface being PtdIns(4,5)P2, the target molecule. Furthermore, this orientation would also explain the biochemical results showing that PtdIns(3,4,5)P3 can also bind (although with lower affinity) to this C2 domain (10, 20, 28), because the phosphate group in the C3 of the inositol ring would point to the membrane interface not being directly involved in the protein interaction (see Fig. 1C and the docking model proposed in Fig. 4A).

Fig. 4.
Docking of the PKCα-C2 domain into the membrane surface. (A) The model membrane corresponds to a POPC molecular dynamics simulation with coordinates (Protein Data Bank ID code popc128a). Half of the membrane bilayer is represented (thin sticks). ...

Role of the Aromatic Residues on the Plasma Membrane Localization of PKCα.

In an attempt to correlate the binding properties determined in the crystal structure with the function of Tyr-195 and Trp-245 on the plasma membrane localization of PKCα, we performed site-directed mutagenesis with the full-length PKCα fused to EGFP (PKCα-EGFP). To abrogate the interactions exerted by these residues with the PtdIns(4,5)P2 molecule, several mutants were generated: PKCαW245A-EGFP, PKCαY195S-EGFP, PKCαY195S/W245A-EGFP, PKCαK209A/K211A/Y195S-EGFP, and PKCαK209A/K211A/Y195S/W245A-EGFP. The cell system used was neural growth factor (NGF)-differentiated pheochromocytoma cells (PC12) that were stimulated with extracellular ATP to induce the plasma membrane translocation of PKCα (17).

When PKCαW245A-EGFP and PKCαY195S-EGFP mutants were tested in differentiated PC12 cells under the same conditions as the WT PKCα, it was observed that only 31% and 43% of the cells expressing these constructs, respectively, were able to exhibit partial plasma membrane. Their t1/2 was not affected significantly but their half-maximal dissociation times (HMDT) decreased ≈50% (Table 1), suggesting that both Trp-245 and Tyr-195 are important in the membrane docking of PKCα. When the cells were stimulated with ionomycin and 1,2-dioctanoyl-sn-glycerol (DiC8), a higher number of cells responded to the stimulation, although the membrane/localization ratio was very similar to the ATP stimulation and the half-time of translocation was slower than that obtained for the WT protein under the same conditions (Fig. 2 A and B and Table 1), demonstrating that even at saturating Ca2+ and diacylglycerol concentrations, the mutant proteins were not able to properly localize in the plasma membrane. Very similar results were obtained with the PKCαY195S/W245A-EGFP double mutant that now appeared localized (only 34%) in vesicles spread all over the cytosol, suggesting that the mutations impede PKCα to find its target in the plasma membrane and, calcium and diacylglycerol excess cannot recover its proper localization in the membrane (Fig. 2 C and D and Table 1).

Table 1.
Plasma membrane translocation parameters calculated for the different mutants studied
Fig. 2.
Role of aromatic residues located in the polybasic cluster on PKCα membrane localization. (A) Confocal images of dPC12 cells expressing PKCα-EGFP and stimulated with 100 μM ATP. The left and center frames correspond to 0 and 60 ...

Because the Lys residues located in the β4 strand establish interactions with 2 phosphate moieties, we studied the effect of triple and quadruple mutations including these residues (Lys-209 and Lys-211), Tyr-195 and Trp-245, to explore their effect on the localization of the enzyme. It was observed that both mutants reduced their ability to bind to the plasma membrane and were also found in vesicles spread over the cytosol (Table 1 and Fig. S3).

Both structural and functional results suggest that aromatic and cationic residues are involved in the PtdIns(4,5)P2 recognition by interacting with the oxygen moieties of phosphates 1, 4, and 5 of the inositide ring. Taking into account that mutations of the residues forming this region do not affect the apparent Ca2+ affinity of the C2 domain (19, 23), the effect observed on the plasma membrane localization seems to be derived mainly from the reduced affinity of these mutants for PtdIns(4,5)P2.

The Dominant Interfering Activity of the C2 Domain Is Blocked by Abolishing All of the Residues Involved in the PtdIns(4,5)P2 Binding.

We demonstrated in previous work that the isolated C2 domain of PKCα, when overexpressed in PC12 cells, acts as a dominant negative protein module that inhibits the neuronal differentiation induced by NGF and ATP in these cells (21). Here, we tested the effect of several mutants on the neuronal differentiation process induced by NGF and ATP for 48 h. The results show that C2αW245A-enhanced cyan fluorescent protein (ECFP), C2αY195A-ECFP, C2αY195A/W245A-ECFP, and C2αK209A/K211A-ECFP mutants partially recovered the neuronal differentiation (Fig. 2E and Table S2). Only when the 4 mutations were included into the same construct (C2αK209A/K211A/Y195S/W245A-ECFP) was a total recovery of the neuronal differentiation obtained, suggesting that once the 4 critical sites are eliminated the domain lacks its ability to bind PtdIns(4,5)P2 in the plasma membrane and consequently does not interfere in this long-term process. Note that the experiments performed with the same mutant in full-length protein by stimulating PC12 cells with ATP showed that PKCα was still able to transiently interact with the plasma membrane, probably because of the cooperation of the calcium-binding region that is still able to interact with PtdSer, but not able to interfere in the differentiation process.

The Aromatic and Cationic Residues in the β3–β4 Groove Are Highly Conserved Among C2 Domains of Topology I.

We analyzed the similarity of the C2 domains of cPKCs with other C2 domains of topology I and II by structure-based sequence alignment using VAST-MMDB, National Center for Biotechnology Information Structure Group (29, 30). It was observed that Tyr-195 in PKCα is very well conserved in all of them (Fig. S4). Lys-197, Lys-209, and Asn-253 are highly conserved in domains with topology I, and Lys-211 is also conserved in domains with topology I except in the C2A domain of the synaptotagmins analyzed, and the C2A domain of DOC2γ (Fig. S4). Finally, Trp-245 is conserved in most C2 domains of topology I or substituted by residues like Leu, Cys, or Tyr. However, the C2 domains of topology II do not conserve most of the residues responsible for the PtdIns(4,5)P2 interaction (Fig. S4).

Together, these results suggest that the existence of these amino acidic residues in the primary sequence of a C2 domain could enable us to predict that there is a potential PtdIns(4,5)P2 interacting site with the following consensus sequence: Tyr X3 Lys Xn1 LysXLys Xn2 Trp (Tyr/Leu/Cys) Xn3 Asn, where Xn1 represents the connecting loop between β3 and β4 and Xn2 corresponds to a segment including β5, β6, and their connecting loops. This could imply that the number of amino acidic residues can vary in each particular C2 domain (Fig. S4).

Because the Trp-245 residue was the less conserved among these C2 domains aligned, we wondered whether in PKCα this residue could be substituted by Leu or Tyr. It was observed that the mutation to Leu inhibited the plasma membrane localization in 50% of the cells analyzed (Fig. S5A). However, the mutation to Tyr produced no significant changes in the plasma membrane localization parameters measured (Fig. S5 B and C), suggesting that in the case of PKCα, the hydroxyl group of Tyr is able to establish the hydrogen bond with phosphate 5 in the inositol ring.

The highest scores obtained by the structure-based sequence alignment (VAST) corresponded to the C2 domains of synaptotagmin I and VII, rabphilin 3A and PI3KC2α. All of them have been described to bind PtdIns(4,5)P2 (3, 4, 31, 32), and they conserved most of the residues included in the consensus site (Fig. S4). When the 3D alignment structures of synaptotagmin I-C2B and PKCα-C2 were compared (Fig. 3 A and B), the former conserves most of the essential residues for PtdIns(4,5)P2 binding (Fig. 3A). However, these residues have not been studied in synaptotagmins (Fig. 3B), and their contribution to neuronal exocytosis will have to be further explored.

Fig. 3.
Structural superimpositions of the C2 domain of PKCα with other C2 domains of topology I. The region represented contains all residues of PKCα C2 involved in direct contacts with PtdIns(4,5)P2 (dark blue) and the equivalent residues in ...

The C2A and C2B domains of rabphilin 3A and the C2 domain of PI3KC2α also bind PtdIns(4,5)P2 (3, 4, 32) with different affinities, and they conserve most of the residues described in PKCα (Fig. 3 C–E). It is interesting to note that the C2B domain of rabphilin 3A exhibits very low affinity to bind PtdIns(4,5)P2 (4), and when its 3D structure was overlapped with those of PKCα it was observed that Tyr-195 in PKCα is substituted by Phe-579 in rabphilin 3A-C2B (Fig. 3D). This subtle change might impede the formation of 2 H bonds between Phe-579 and phosphates 4 and 5 of PtdIns(4,5)P2, thus explaining why the C2B domain exhibits a very low binding affinity and confirming that a Tyr residue is necessary in this position.

Model for Membrane Docking of the PKCα-C2 Domain.

cPKCs are kinases involved in a wide variety of signaling processes. The results obtained in this work allowed us to identify the molecular determinants that control the PtdIns(4,5)P2-C2 domain interaction, showing an unexpected mechanism of interaction with the plasma membrane because aromatic and cationic residues are able to interact directly with the PtdIns(4,5)P2 moieties (Fig. 4). Another relevant finding in this work is the demonstration that, at least, the C2 domain of PKCα interacts specifically with PtdSer and PtdIns(4,5)P2 through 2 independent motifs (Fig. 4), which enables the domain to be anchored in the membrane by 2 points, which is essential for its proper function in the membrane interface. Finally, a consensus site for PtdIns(4,5)P2-binding (polybasic region or β3–β4 groove) has been determined. This site exists not only in the C2 domains of cPKCs but also in a wide variety of other C2 domains of topology I like synaptotagmin, rabphilin 3A, and PI3KC2α. Because of the important functions played by these other proteins in neuronal transmission, vesicle fusion, or cell signaling, further studies will be needed to shed light on the role of PtdIns(4,5)P2 in the cellular processes in which they participate.

Materials and Methods

Protein Purification and Crystallization of Complexes.

The recombinant PKCα C2 domain (residues from His-155 to Gly-293) was obtained and purified as described (11). Two different complexes were obtained. The C2 domain at 4 mg/mL was incubated overnight at 4 °C with 25 mM CaCl2 and with either 2 mM PtdIns(4,5)P2 or 2 mM PtdSer and 2 mM PtdIns(4,5)P2. Crystals were grown by using the hanging drop vapor diffusion method in the conditions as described (11, 16, 22). Data collection and refinement statistic are shown in Table S1.

More Methods.

Additional details are in SI Text and Fig. S6.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Mònica Buxaderas for obtaining crystals. Work in Murcia was supported by the Fundación Médica Mutua Madrileña, Fundación Ramón Areces, and Fundación Séneca 08700/PI/08 (to S.C.-G.), and Ministerio de Ciencia e Innovación Grants BFU2005-02482 and BFU2008-01010 (to J.G.-F.). Work in Barcelona was supported by Ministerio de Ciencia e Innovación Grants BFU2005-02376/BMC (to N.V.) and BFU2005-08686-C02-01 (to I.F.). X-ray data were collected at the European Molecular Biology Laboratory protein crystallography beam line ID14.2 at the European Synchrotron Radiation Facility (Grenoble) within a Block Allocation Group (Barcelona). Financial support was provided by the European Synchrotron Radiation Facility.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3GPE).

This article contains supporting information online at www.pnas.org/cgi/content/full/0813099106/DCSupplemental.

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