Ca2+ bridges the C2 membrane-binding domain of protein kinase Cα directly to phosphatidylserine

Abstract

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.

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…The Protein Data Bank (Berman et al, 2000) identifiers for the experimentally determined C2 domain models in Figure 1 are 1RLW (cPLA 2␣ C2; Perisic et al, 1998), and 1DSY (PKC␣C2; Verdaguer et al, 1999). The hybrid C2 domain models illustrated in Figure 1A were constructed by placing the respective CBL and ␤3-4 regions from PKC␣C2 onto the structural core of cPLA 2 C2.…”

“…Because the crystal structures for cPLA 2 ␣C2 and PKC␣C2 are known (Perisic et al, 1998;Verdaguer et al, 1999), it was possible to align homologous structures to produce the hybrids ( Figure 1B). Direct comparison of targeting between the cPLA 2 /PKC_CBLs hybrid fused to CFP (CFP-cPLA 2 /PKC_CBLs) and YFP-PKC␣C2 revealed Ca 2ϩ -dependent translocation of the hybrid from the cytoplasm to a juxtanuclear location, with some binding to the plasma membrane still detectable (Figure 6A and Supplementary Figure 6Avideo).…”

“…Ca 2ϩ binding in the pocket defined by the three CBLs of the PKC␣ C2 domain has long been recognized as critical for the translocation of PKC␣ and the isolated PKC␣ C2 domain to the plasma membrane in cells and for lipid bilayer binding in vitro (Medkova and Cho, 1998;Corbalan-Garcia et al, 1999;Verdaguer et al, 1999;Conesa-Zamora et al, 2001;Stahelin and Cho, 2001;Kohout et al, 2002Kohout et al, , 2003Murray and Honig, 2002). In vitro, and presumably in vivo as well, this translocation involves binding of the Ca 2ϩ -loaded protein to anionic PS headgroups on the membrane surface (Verdaguer et al, 1999;(Evans et al, 2004).…”

“…The PKC␣ C2 domain is structurally characterized by eight antiparallel ␤ strands connected by interstrand loops to form a beta-sandwich structure (see Figure 1A; Verdaguer et al, 1999). After increases in intracellular Ca 2ϩ concentrations, the PKC␣ C2 domain binds 2 Ca 2ϩ ions in a pocket defined by three interstrand loops (Ca 2ϩ -binding loops or CBLs), leading to a favorable electrostatic interaction with the isolated headgroup of the anionic lipid phosphatidylserine (PS) (Verdaguer et al, 1999) and with PScontaining membranes (Murray and Honig, 2002;Evans et al, 2004). Previous studies have concluded that the CBLs are primarily or fully responsible for specific targeting to the plasma membrane in preference to other intracellular membranes (Stahelin et al, 2003;Marin-Vicente et al, 2005).…”

“…Previous studies have concluded that the CBLs are primarily or fully responsible for specific targeting to the plasma membrane in preference to other intracellular membranes (Stahelin et al, 2003;Marin-Vicente et al, 2005). In addition to the CBLs, a second membrane-interacting region consisting of basic residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been identified and shown to interact with PS or another membrane lipid, PIP 2 (Verdaguer et al, 1999;Ochoa et al, 2002;Corbalan-Garcia et al, 2003). Mutations at key positions in the CBLs or in the ␤3-4 hairpin interfere with plasma membrane interaction in vivo and reduce the affinity of the PKC␣ C2 domain for PS or PIP 2 in vitro (Medkova and Cho, 1998;Ochoa et al, 2002;Bolsover et al, 2003;Stahelin et al, 2003;Rodriguez-Alfaro et al, 2004;Marin-Vicente et al, 2005).…”