Structural and Mechanistic Insights Into the Association of PKC -C2 Domain to Ptdins(4,5)

Structural and mechanistic insights into the association of PKC␣-C2 domain to PtdIns(4,5)P2 Marta Guerrero-Valeroa,1, Cristina Ferrer-Ortab,1, Jordi Querol-Audíb, Consuelo Marin-Vicentea, Ignacio Fitab,c, Juan C. Go´ mez-Ferna´ ndeza, Nuria Verdaguerb,2, and Senena Corbala´ n-Garcíaa,2

aDepartment of Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain; bInstituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Cientiﬁcas, Parque Cientíﬁco de Barcelona, and cInstitute for Research in Biomedicine, Baldiri i Reixac 10, 08028 Barcelona, Spain

Edited by John Kuriyan, University of California, Berkeley, CA, and approved February 27, 2009 (received for review December 22, 2008)

C2 domains are widely-spread protein signaling motifs that in classical in the PtdIns(4,5)P2-C2 domain interaction. Noteworthy, most of PKCs act as Ca2؉-binding modules. However, the molecular mecha- the residues found in the phosphoinositide interaction are highly nisms of their targeting process at the plasma membrane remain conserved among C2 domains of topology I, and a general poorly understood. Here, the crystal structure of PKC␣-C2 domain in mechanism for membrane docking of C2 domains regulated 2؉ complex with Ca , 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] specifically by PtdIns(4,5)P2 is proposed. (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P2] shows that PtdSer binds speciﬁcally to the Results and Discussion calcium-binding region, whereas PtdIns(4,5)P2 occupies the con- PtdIns(4,5)P2 Binds Specifically to the ␤3–␤4 Groove in the PKC␣-C2 cave surface of strands ␤3 and ␤4. Strikingly, the structure reveals Domain. To understand how the C2 domain of cPKCs interacts a PtdIns(4,5)P2-C2 domain-binding mode in which the aromatic with its 2 targets: PtdSer and PtdIns(4,5)P2 2 different crystals residues Tyr-195 and Trp-245 establish direct interactions with the forms were obtained of the recombinant PKC␣-C2 domain. 2ϩ phosphate moieties of the inositol ring. Mutations that abrogate One crystallized in the presence of Ca and PtdIns(4,5)P2, 2ϩ Tyr-195 and Trp-245 recognition of PtdIns(4,5)P2 severely impaired and the other crystallized in the presence of Ca , ␣ the ability of PKC to localize to the plasma membrane. Notably, PtdIns(4,5)P2, and PtdSer. The final electron density maps these residues are highly conserved among C2 domains of topology confirmed the presence of the phospholipid ligands in both I, and a general mechanism of C2 domain-membrane docking medi- structures (Fig. 1 and Table S1). 2ϩ ated by PtdIns(4,5)P2 is presented. The3Ca ions found at the calcium-binding pocket in the 2 complexes are located in equivalent positions to the calcium sites calcium phosphoinositides ͉ peripheral membrane proteins Ca1, Ca2 ,and Ca3, as described (16, 22) and in Fig. S1. In the 2ϩ PKC␣C2-Ca -PtdIns(4,5)P2 structure, a strong peak of extra he C2 domains are considered peripheral proteins that are electron density in this region was interpreted by the presence of Twater-soluble and associate reversibly with lipid bilayers. a phosphate ion, completing the coordination of Ca1 (Fig. S1A). ␣ 2ϩ Recently, evidence has demonstrated that some of these do- In the PKC C2-Ca -PtdSer-PtdIns(4,5)P2 structure, a more mains are able to interact with the inositol phospholipid 1,2- elongated density appeared close to Ca1 that was interpreted as diacyl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] corresponding to a partially-ordered phosphoserine head group [PtdIns(4,5)P2] (1–4), which is able to directly participate in a of PtdSer (Fig. S1B). myriad of functions, including cell signaling at the plasma Well-defined extra electron densities were found, in both membrane, regulation of membrane traffic and transport, cy- complexes, within the concave surface formed by strands ␤3 and toskeleton dynamics, and nuclear events (5, 6). Despite the ␤4 of the C2 domain, the ␤3–␤4 groove. These densities were number of C2 domain 3D structures currently available, ques- clearly explained by the presence of the inositol(1,4,5)- tions about how they interact with the different target phospho- trisphosphate (InsP3) head groups of PtdIns(4,5)P2, occupying lipids, their precise spatial position in the lipid bilayer, and their the cavity (Fig. 1 A and B). When the InsP3 head group of role in transmitting signals downstream have yet to be explored. PtdIns(4,5)P2 is positioned in the region, all 3 phosphate groups The main role of the C2 domain in classical PKCs (cPKCs) is and, to a lesser extent, the polyalcohol moiety interact with the to act as the Ca2ϩ-activated membrane-targeting motif (7, 8). C2 domain (Fig. 1). Three of the 4 conserved lysines within the The 3D structure of these C2 domains comprises 8 antiparallel polybasic cluster are involved in interactions with PtdIns(4,5)P2: ␤-strands assembled in a ␤-sandwich architecture, with flexible Lys-197 of ␤3 forms 2 polar bonds with the phosphate 5 moiety loops on top and at the bottom (9–12). This C2 domain displays and the hydroxyl group O6 of the inositol ring, Lys-209 of ␤4 BIOCHEMISTRY 2 functional regions: the Ca2ϩ-binding region and the polybasic forms a salt bridge with the phosphate 1 that remains partially cluster. The former is located in the flexible top loops, binds 2 exposed to the solvent, and Lys-211, also from ␤4, interacts with or3Ca2ϩ ions, depending on the isoenzyme (10, 11, 13, 14), and the phosphate 4 moiety. In addition, 3 other residues participate interacts with 1,2-diacyl-sn-glycero-3-[phospho-L-serine] (Ptd- Ser) (11, 15, 16). The second region is a polybasic cluster that is Author contributions: N.V. and S.C.-G. designed research; M.G.-V., C.F.-O., J.Q.-A., C.M.-V., located at the concave surface of the C2 domain formed by N.V., and S.C.-G. performed research; M.G.-V., C.F.-O., J.Q.-A., C.M.-V., N.V., and S.C.-G. strands ␤3 and ␤4. Recent studies indicate that this region might analyzed data; and I.F., J.C.G.-F., N.V., and S.C.-G. wrote the paper. 2ϩ bind specifically to PtdIns(4,5)P2 inaCa -dependent manner The authors declare no conﬂict of interest. (1, 17–21). This article is a PNAS Direct Submission. To gain insight into the structural and functional basis for the Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, PtdIns(4,5)P2-dependent membrane targeting of the PKC␣-C2 www.pdb.org (PDB ID code 3GPE). domain, we determined the 3D structures of the ternary and 1M.G.-V. and C.F-.O. contributed equally to this work. ␣ quaternary complexes of the C2 domain of PKC , crystallized in 2To whom correspondence may be addressed. E-mail: [email protected] or 2ϩ 2ϩ presence of Ca and PtdIns(4,5)P2 or Ca , PtdIns(4,5)P2 and [email protected]. PtdSer. In addition, the crystallographic results were validated in This article contains supporting information online at www.pnas.org/cgi/content/full/ living cells by site-directed mutagenesis of the residues involved 0813099106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813099106 PNAS ͉ April 21, 2009 ͉ vol. 106 ͉ no. 16 ͉ 6603–6607 Downloaded by guest on September 28, 2021 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).

Role of the Aromatic Residues on the Plasma Membrane Localization of PKC␣. 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 PKC␣,we performed site-directed mutagenesis with the full-length PKC␣ fused to EGFP (PKC␣-EGFP). To abrogate the interactions exerted by these residues with the PtdIns(4,5)P2 molecule, several mutants were generated: PKC␣W245A-EGFP, PKC␣Y195S-EGFP, PKC␣Y195S/W245A-EGFP, PKC␣K209A/ K211A/Y195S-EGFP, and PKC␣K209A/K211A/Y195S/ W245A-EGFP. The cell system used was neural growth factor (NGF)-differentiated pheochromocytoma cells (PC12) that ␣ ␣ Fig. 1. Structure of PKC C2 domain bound to PtdIns(4,5)P2.(A) The PKC C2- were stimulated with extracellular ATP to induce the plasma 2ϩ ␣ 2ϩ Ca -PtdIns(4,5)P2 ternary complex. (B) The PKC C2-Ca -PS-PtdIns(4,5)P2 membrane translocation of PKC␣ (17). quaternary complex. The C2 molecule is shown in blue. The calcium ions When PKC␣W245A-EGFP and PKC␣Y195S-EGFP mutants located at the tip of the domain in the calcium-binding regions are repre- were tested in differentiated PC12 cells under the same condi- sented as green spheres. The phosphate ion and the phospholipids molecules ␣ are depicted as sticks. (C) View of the ␤3–␤4 groove, showing the interactions tions as the WT PKC , it was observed that only 31% and 43% between the C2 domain and the IP3 headgroup of PtdIns(4,5)P2. The C2 of the cells expressing these constructs, respectively, were able to residues are represented as blue sticks and explicitly labeled. The InsP3 mol- exhibit partial plasma membrane. Their t1/2 was not affected ecule is shown in sticks in atom type code. Hydrogen bonds are shown as significantly but their half-maximal dissociation times (HMDT) Խ ԽϪԽ Խ dashed lines in black. An omit Fo Fc electron density map, contoured at 3.0 decreased Ϸ50% (Table 1), suggesting that both Trp-245 and ␴ , is shown in green around the InsP3 molecule. Tyr-195 are important in the membrane docking of PKC␣. When the cells were stimulated with ionomycin and 1,2-dioctanoyl-sn- in the interaction: the hydroxyl group of Tyr-195 is hydrogen- glycerol (DiC8), a higher number of cells responded to the bonded to phosphates 4 and 5. The side chain of Asn-253 forms stimulation, although the membrane/localization ratio was very a weak hydrogen bond with the phosphate 5 moiety (distance similar to the ATP stimulation and the half-time of translocation 3.51 Å). Finally, Trp-245, located in the proximity, also contacts was slower than that obtained for the WT protein under the same phosphate 5 (Fig. 1C). These findings are in accordance with the conditions (Fig. 2 A and B and Table 1), demonstrating that even 2ϩ biochemical results obtained previously, because mutations at saturating Ca and diacylglycerol concentrations, the mutant abolishing the calcium-binding region affected only the PtdSer proteins were not able to properly localize in the plasma binding (15, 23, 24), and mutations abolishing the concave region membrane. Very similar results were obtained with the ␣ formed by strands ␤3 and ␤4 altered only the PtdIns(4,5)P2 PKC Y195S/W245A-EGFP double mutant that now appeared interaction (1, 16, 19). In addition, the interaction between localized (only 34%) in vesicles spread all over the cytosol, aromatic residues and phosphoinositide head groups has also suggesting that the mutations impede PKC␣ to find its target in been observed in other peripheral membrane proteins like some the plasma membrane and, calcium and diacylglycerol excess pleckstrin homology domains (25). cannot recover its proper localization in the membrane (Fig. 2 The results obtained above immediately suggested that an C and D and Table 1). ␤ InsP3 molecule would also bind to the C2 domain in a similar way Because the Lys residues located in the 4 strand establish to the PtdIns(4,5)P2 molecule. Because of the importance of interactions with 2 phosphate moieties, we studied the effect InsP3 in the signal transduction pathways involving PKC␣ acti- of triple and quadruple mutations including these residues vation, we examined the thermodynamics of soluble InsP3 bind- (Lys-209 and Lys-211), Tyr-195 and Trp-245, to explore their ing to the C2 domain of PKC␣ by isothermal titration calorim- effect on the localization of the enzyme. It was observed that etry at 25 °C in the presence of saturating Ca2ϩ. The binding data both mutants reduced their ability to bind to the plasma demonstrated that there is 1 binding site but that is not com- membrane and were also found in vesicles spread over the pletely occupied (n ϭ 0.51 Ϯ 0.14) with a KD of 19.8 Ϯ 1.7 ␮M cytosol (Table 1 and Fig. S3). (Fig. S2). These results demonstrate that the affinity of the C2 Both structural and functional results suggest that aromatic domain for PtdIns(4,5)P2 (KD ϭ 1.8 ␮M in ref. 19) is higher than and cationic residues are involved in the PtdIns(4,5)P2 recogni- for soluble InsP3 or PtdSer (KD ϭ 18 ␮M in ref. 20), suggesting tion by interacting with the oxygen moieties of phosphates 1, 4, that other phosphoinositide moieties and/or the orientation and 5 of the inositide ring. Taking into account that mutations adopted by the PtdIns(4,5)P2 molecule in the membrane might of the residues forming this region do not affect the apparent contribute to increase this affinity. Ca2ϩ affinity of the C2 domain (19, 23), the effect observed on Several studies have demonstrated that the unphosphorylated the plasma membrane localization seems to be derived mainly inositol ring is oriented perpendicular to the bilayer surface (26), from the reduced affinity of these mutants for PtdIns(4,5)P2.

6604 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813099106 Guerrero-Valero et al. Downloaded by guest on September 28, 2021 Table 1. Plasma membrane translocation parameters calculated for the different mutants studied Stimulation

100 ␮M ATP Ionomycin ϩ 100 ␮g/mL DiC8

Membrane Membrane

Protein N cells localization, % Rmax,% t1/2, s HMDT, s localization, % Rmax,% t1/2,s

WT PKC␣-EGFP 37 70 0.74 Ϯ 0.13 12 Ϯ 3 130 Ϯ 28 100 0.83 Ϯ 0.09 13 Ϯ 7 PKC␣W245A-EGFP 19 31 0.52 Ϯ 0.14 16 Ϯ 457Ϯ 33 84 0.62 Ϯ 0.11 38 Ϯ 16 PKC␣Y195S-EGFP 23 43 0.63 Ϯ 0.11 13 Ϯ 469Ϯ 19 70 0.57 Ϯ 0.12 57 Ϯ 13 PKC␣Y195S/W245A-EGFP 29 34 0.45 Ϯ 0.11 25 Ϯ 785Ϯ 30 55 0.49 Ϯ 0.16 42 Ϯ 19 PKC␣K209A/K211A-EGFP 26 40 0.55 Ϯ 0.12 22 Ϯ 863Ϯ 29 50 0.40 Ϯ 0.1 64 Ϯ 25 PKC␣K197A/K199A-EGFP 20 55 0.57 Ϯ 0.14 18 Ϯ 484Ϯ 27 50 0.30 Ϯ 0.06 32 Ϯ 15 PKC␣K209A/K211A/Y195S-EGFP 31 45 0.52 Ϯ 0.13 25 Ϯ 783Ϯ 28 36 0.32 Ϯ 0.08 39 Ϯ 29 PKC␣K209A/K211A/Y195S/W245A-EGFP 28 42 0.42 Ϯ 0.1 23 Ϯ 786Ϯ 10 39 0.40 Ϯ 0.12 59 Ϯ 19

Membrane localization indicates the percentage of cells responding to ATP stimulation with plasma membrane translocation. Rmax is the maximal relative increase in plasma membrane localization of the enzyme. t1/2 is the half-time of translocation. HMDT is the half-maximal dissociation time.

The Dominant Interfering Activity of the C2 Domain Is Blocked by interact with the plasma membrane, probably because of the Abolishing All of the Residues Involved in the PtdIns(4,5)P2 Binding. cooperation of the calcium-binding region that is still able to We demonstrated in previous work that the isolated C2 domain interact with PtdSer, but not able to interfere in the differenti- of PKC␣, when overexpressed in PC12 cells, acts as a dominant ation process. negative protein module that inhibits the neuronal differentiation induced by NGF and ATP in these cells (21). Here, we tested The Aromatic and Cationic Residues in the ␤3–␤4 Groove Are Highly the effect of several mutants on the neuronal differentiation Conserved Among C2 Domains of Topology I. We analyzed the process induced by NGF and ATP for 48 h. The results show that similarity of the C2 domains of cPKCs with other C2 domains of C2␣W245A-enhanced cyan fluorescent protein (ECFP), topology I and II by structure-based sequence alignment using C2␣Y195A-ECFP, C2␣Y195A/W245A-ECFP, and C2␣K209A/ VAST-MMDB, National Center for Biotechnology Information K211A-ECFP mutants partially recovered the neuronal differ- Structure Group (29, 30). It was observed that Tyr-195 in PKC␣ entiation (Fig. 2E and Table S2). Only when the 4 mutations is very well conserved in all of them (Fig. S4). Lys-197, Lys-209, were included into the same construct (C2␣K209A/K211A/ and Asn-253 are highly conserved in domains with topology I, Y195S/W245A-ECFP) was a total recovery of the neuronal and Lys-211 is also conserved in domains with topology I except differentiation obtained, suggesting that once the 4 critical sites in the C2A domain of the synaptotagmins analyzed, and the C2A are eliminated the domain lacks its ability to bind PtdIns(4,5)P2 domain of DOC2␥ (Fig. S4). Finally, Trp-245 is conserved in in the plasma membrane and consequently does not interfere in most C2 domains of topology I or substituted by residues like this long-term process. Note that the experiments performed Leu, Cys, or Tyr. However, the C2 domains of topology II do not with the same mutant in full-length protein by stimulating PC12 conserve most of the residues responsible for the PtdIns(4,5)P2 cells with ATP showed that PKC␣ was still able to transiently interaction (Fig. S4).

ATP I + DiC8 0.8 A Rmax 0.6

0.4 HMDT E ATP

I+DiC8 )% 0.2 80 B ( 0s 60s 350s 0.0 sllec 60 0 100 200 300 400 t1/2

d PKC -EGFP BIOCHEMISTRY

etaitner 40 0.8 C

0.6 effiD D 20 0.4 ATP I+DiC8 0.2 0 0s 60s 350s 123456789 0.0 PKC Y195S/W245A-EGFP 0 100 200 300 400 Time (s)

Fig. 2. Role of aromatic residues located in the polybasic cluster on PKC␣ membrane localization. (A) Confocal images of dPC12 cells expressing PKC␣-EGFP and stimulated with 100 ␮M ATP. The left and center frames correspond to 0 and 60 s after ATP stimulation. PC12 cells were also stimulated with 2 ␮M ionomycin and 100 ␮g/mL DiC8 (right frame) after 300 s of recording. (B) The red arrows indicate how the Rmax and HMDT parameters were calculated; the green arrow indicates how the t1/2 parameter was calculated. The proﬁle is representative of the results obtained in the cells analyzed (n ϭ 37). (C and D) The PKC␣Y195S/W245A-EGFP mutant was studied under the same conditions as the WT protein in A and B (n ϭ 29 cells). (E) PC12 cells were transfected with PKC␣-EGFP (lane 1), C2␣-ECFP (lane 2), C2␣W245A-ECFP (lane 3), C2␣Y245A-ECFP (lane 4), C2␣Y245A/W245A-ECFP (lane 5), C2␣K209A/K211A-ECFP (lane 6), C2␣K209A/K211A/Y195S/W245A-ECFP (lane 7), PKC␣K209A/K211A/Y195S/W245A-EGFP (lane 8), or ECFP-PH-PLC␦ (lane 9) as a control.

Guerrero-Valero et al. PNAS ͉ April 21, 2009 ͉ vol. 106 ͉ no. 16 ͉ 6605 Downloaded by guest on September 28, 2021 Fig. 3. Structural superimpositions of the C2 domain of PKC␣ with other C2 domains of topology I. The region represented contains all residues of PKC␣ C2 involved in direct contacts with PtdIns(4,5)P2 (dark blue) and the equivalent residues in the compared structures (light blue and labeled) of synaptotagmin I-C2B (A and B), rabphilin 3A C2A and C2B domains (C and D), and PI3KC2␣ C2 (E). B shows in yellow sticks the Lys residues mutated in synaptotagmin I-C2B (2, 35, 36); note that Lys-311 and Lys-326 are not homologues to the conserved residues that interact with PtdIns(4,5)P2 in PKC␣ and only Lys-327 in synaptotagmin I-C2B is homologue to Lys-211 in PKC␣.

Together, these results suggest that the existence of these tion, showing an unexpected mechanism of interaction with the amino acidic residues in the primary sequence of a C2 domain plasma membrane because aromatic and cationic residues are could enable us to predict that there is a potential PtdIns(4,5)P2 able to interact directly with the PtdIns(4,5)P2 moieties (Fig. 4). interacting site with the following consensus sequence: Tyr X3 Another relevant finding in this work is the demonstration that, Lys Xn1 LysXLys Xn2 Trp (Tyr/Leu/Cys) Xn3 Asn, where Xn1 at least, the C2 domain of PKC␣ interacts specifically with represents the connecting loop between ␤3 and ␤4 and Xn2 PtdSer and PtdIns(4,5)P2 through 2 independent motifs (Fig. 4), corresponds to a segment including ␤5, ␤6, and their connecting which enables the domain to be anchored in the membrane by loops. This could imply that the number of amino acidic residues 2 points, which is essential for its proper function in the 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 PKC␣ 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 PKC␣, 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 PI3KC2␣. 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 PKC␣-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. The C2A and C2B domains of rabphilin 3A and the C2 domain of PI3KC2␣ also bind PtdIns(4,5)P2 (3, 4, 32) with different affinities, and they conserve most of the residues described in PKC␣ (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 PKC␣ it was observed that Tyr-195 in PKC␣ 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 Fig. 4. Docking of the PKC -C2 domain into the membrane surface. (A) The C2B domain exhibits a very low binding affinity and confirming model membrane corresponds to a POPC molecular dynamics simulation with that a Tyr residue is necessary in this position. coordinates (Protein Data Bank ID code popc128a). Half of the membrane bilayer is represented (thin sticks). The C2 domain in represented as its poten- ␣ tial surface as computed by GRASP (33) and displayed by PyMol (www. Model for Membrane Docking of the PKC -C2 Domain. cPKCs are pymol.org). Ca2ϩ ions are shown as green spheres. The head groups of the 2 kinases involved in a wide variety of signaling processes. The interacting phospholipids, PtdSer and PtdIns(4,5)P2 (thick sticks), served as results obtained in this work allowed us to identify the molecular reference for the docking model. (B) Close-up of the PtdSer and PtdIns(4,5)P2 determinants that control the PtdIns(4,5)P2-C2 domain interac- interacting surfaces.

6606 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813099106 Guerrero-Valero et al. Downloaded by guest on September 28, 2021 membrane interface. Finally, a consensus site for PtdIns(4,5)P2- mM PtdIns(4,5)P2 or 2 mM PtdSer and 2 mM PtdIns(4,5)P2. Crystals were grown binding (polybasic region or ␤3–␤4 groove) has been deter- by using the hanging drop vapor diffusion method in the conditions as mined. This site exists not only in the C2 domains of cPKCs but described (11, 16, 22). Data collection and refinement statistic are shown in Table S1. also in a wide variety of other C2 domains of topology I like ␣ synaptotagmin, rabphilin 3A, and PI3KC2 . Because of the More Methods. Additional details are in SI Text and Fig. S6. important functions played by these other proteins in neuronal transmission, vesicle fusion, or cell signaling, further studies will ACKNOWLEDGMENTS. We thank Mo`nica Buxaderas for obtaining crystals. Work in Murcia was supported by the Fundacioń Me´ dica Mutua Madrilenã, be needed to shed light on the role of PtdIns(4,5)P2 in the Fundacioń Ramoń Areces, and FundaciońSe´ neca 08700/PI/08 (to S.C.-G.), and cellular processes in which they participate. Ministerio de Ciencia e Innovacioń Grants BFU2005-02482 and BFU2008-01010 (to J.G.-F.). Work in Barcelona was supported by Ministerio de Ciencia e Materials and Methods Innovacioń Grants BFU2005-02376/BMC (to N.V.) and BFU2005-08686-C02-01 Protein Purification and Crystallization of Complexes. The recombinant PKC␣ (to I.F.). X-ray data were collected at the European Molecular Biology Labo- ratory protein crystallography beam line ID14.2 at the European Synchrotron C2 domain (residues from His-155 to Gly-293) was obtained and purified as Radiation Facility (Grenoble) within a Block Allocation Group (Barcelona). described (11). Two different complexes were obtained. The C2 domain at 4 Financial support was provided by the European Synchrotron Radiation mg/mL was incubated overnight at 4 °C with 25 mM CaCl2 and with either 2 Facility.

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