N.Verdaguer and S.Corbalan-Garcia contributed equally to this workThe C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca 2⍣ -bound forms of the PKCα-C2 domain both in the absence and presence of 1,2-dicaproyl-snphosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 Å resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca 2⍣ ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca 2ϩ binding region. The central feature of the PKCα-C2 domain structure is an eight-stranded, antiparallel β-barrel with a molecular topology and organization of the Ca 2⍣ binding region closely related to that found in PKCβ-C2, although only two Ca 2⍣ ions have been located bound to the PKCα-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCα-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.

C2 domains are membrane-binding modules that share a common overall fold: a single compact Greek-key motif organized as an eight-stranded anti-parallel β-sandwich consisting of a pair of four-stranded β-sheets. A myriad of studies have demonstrated that in spite of sharing the common structural β-sandwich core, slight variations in the residues located in the interconnecting loops confer C2 domains with functional abilities to respond to different Ca(2+) concentrations and lipids, and to signal through protein-protein interactions as well. This review summarizes the main structural and functional findings on Ca(2+) and lipid interactions by C2 domains, including the discovery of the phosphoinositide-binding site located in the β3-β4 strands. The wide variety of functions, together with the different Ca(2+) and lipid affinities of these domains, converts this superfamily into a crucial player in many functions in the cell and more to be discovered. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

The independently folding C2 domain motif serves as a Ca(2+)-dependent membrane docking trigger in a large number of Ca(2+) signaling pathways. A comparison was initiated between three closely related C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms alpha, beta, and gamma). The results reveal that these C2 domain isoforms exhibit some similarities but are specialized in important ways, including different Ca(2+) stoichiometries. In the absence of membranes, Ca(2+) affinities of the isolated C2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca(2+) binding for the PKC beta C2 domain but not for the PKC alpha or PKC gamma C2 domain (H = 2.3 +/- 0.1 for PKC beta, 0.9 +/- 0.1 for PKC alpha, and 0.9 +/- 0.1 for PKC gamma). When phosphatidylserine-containing membranes are present, Ca(2+) affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca(2+)](1/2) = 0.7 +/- 0.1 microM for PKC gamma, 1.4 +/- 0.1 microM for PKC alpha, and 5.0 +/- 0.2 microM for PKC beta), and cooperative Ca(2+) binding is observed for all three C2 domains (Hill coefficients equal 1.8 +/- 0.1 for PKC beta, 1.3 +/- 0.1 for PKC alpha, and 1.4 +/- 0.1 for PKC gamma). The large effects of membranes are consistent with a coupled Ca(2+) and membrane binding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca(2+). The net negative charge of the phospholipid is more important to membrane affinity than its headgroup structure, although a slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca(2+) stoichiometries of the membrane-bound C2 domains are detectably different. PKC beta and PKC gamma each bind three Ca(2+) ions in the membrane-associated state; membrane-bound PKC alpha binds two Ca(2+) ions, and a third binds weakly or not at all under physiological conditions. Overall, the results indicate that conventional PKC C2 domains first bind a subset of the final Ca(2+) ions in solution, and then associate weakly with the membrane and bind additional Ca(2+) ions to yield a stronger membrane interaction in the fully assembled tertiary complex. The full complement of Ca(2+) ions is needed for tight binding to the membrane. Thus, even though the three C2 domains are 64% identical, differences in Ca(2+) affinity, stoichiometry, and cooperativity are observed, demonstrating that these closely related C2 domains are specialized for their individual functions and contexts.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca 2؉ -binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKC␣-C2 domain in complex with Ca 2؉ , 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 ␤3 and ␤4. 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 PKC␣ 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.calcium phosphoinositides ͉ peripheral membrane proteins T he 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)P 2 ] (1-4), 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 Ca 2ϩ -activated membrane-targeting motif (7, 8). The 3D structure of these C2 domains comprises 8 antiparallel ␤-strands assembled in a ␤-sandwich architecture, with flexible loops on top and at the bottom (9-12). This C2 domain displays 2 functional regions: the Ca 2ϩ -binding region and the polybasic cluster. The former is located in the flexible top loops, binds 2 or 3 Ca 2ϩ 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 ␤3 and ␤4. Recent studies indicate that this region might bind specifically to PtdIns(4,5)P 2 in a Ca 2ϩ -dependent manner (1,(17)(18)(19)(20)(21).To gain insight into the structural and functional basis for the PtdIns(4,5)P 2 -dependent membrane targeting of the PKC␣-C2 domain, we determined the 3D structures of the ternary and quaternary complexes of the C2 domain of PKC␣, crystallized in presence of Ca 2ϩ and PtdIns(4,5...

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) as a second messenger, we studied the activation of protein kinase C␣ by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase C␣, we have determined and characterized the PtdIns(4,5)P 2 -binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysinerich cluster located in the ␤3-and ␤4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P 2 ; and the second, the already well described site formed by the Ca 2؉ -binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase C␣ needs Ca 2؉ before the PtdIns(4,5)P 2 -dependent activation of the enzyme can occur.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected in the plasma membrane but is also found in secretory vesicles, lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus (1-5). PtdIns(4,5)P 2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity, or it can act as a precursor for the generation of different second messengers. For example, several families of phospholipase C enzymes are responsible for the hydrolysis of PtdIns(4,5)P 2 in cells, leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (4, 6), which may, in turn, lead to the activation of different proteins such as some PKC isotypes.Protein kinase C (PKC) composes a large family of serine/ threonine kinases, which is activated by many extracellular signals and plays a critical role in many signal-transducing pathways in the cell (7-9). Based on their enzymatic properties, the mammalian PKC isotypes have been grouped into smaller subfamilies. The first group, which includes the classical isoforms ␣, ␤I, ␤II, and ␥, can be distinguished from the other groups because its activity is regulated by diacylglycerol (DAG) and, cooperatively, by Ca 2ϩ and acidic phospholipids, particularly phosphatidylserine (PS). Members of the second group are the novel mammalian (␦, ⑀, , and ) and yeast PKCs that are not regulated by Ca 2ϩ . The third group comprises the atypical PKC isoforms, , , and , whose regulation has not been clearly established, although it is clear that they are not regulated by DAG or Ca 2ϩ (8, 10). In classical PKC isoenzymes, Ca 2ϩ -dependent binding to membranes shows a high specificity for 1,2-sn-phosphatidyl-Lserine (11)(12)(13)(14). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds, such as arachidonic acid and fre...

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca 2+ -dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Cα (PKCα) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca 2+ binding loops and an anion binding site on β-strands 3-4, such that the β-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca 2+ binding loops and the β-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCα C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca 2+ activation or membrane docking trigger measurable fluorescence changes. Ca 2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca 2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Φ, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca 2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca 2+ first and third Ca 2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on β-strands 3-4 lies near the headgroups. The data support a model in which the β-strands are tilted toward the parallel orientation relative to the membrane surface. † Support provided by NIH Grant GM R01-63235 (to J.J.F.), and by DGESIC Grant PB98-0389 (to J.C.G.F.). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe C2 domain is a conserved membrane docking motif found in numerous eukaryotic signaling proteins. The cellular functions regulated by this ubiquitous signaling domain include: (a) phosphorylation of membrane proteins, (b) production and degradation of lipid derived second messengers, (c) targeting and fusion of vesicles and membranes, (d) G protein signaling by the Ras superfamily, and (e) membrane protein ubiquitination (1-3). While sharing few sequence identities, the structural similarities between different C2 ...

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