Additional Binding Sites for Anionic Phospholipids and Calcium Ions in the Crystal Structures of Complexes of the C2 Domain of Protein Kinase Cα

Abstract

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.

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

“…Domain-containing the PKC␣ C2 Ca 2؉ -binding Loops and the ␤3-4 Hairpin A basic region formed by lysine residues in the ␤-hairpin formed by ␤ strands 3 and 4 has been postulated to coordinate PS or PIP 2 in the membrane (Ochoa et al, 2002;Corbalan-Garcia et al, 2003;Rodriguez-Alfaro et al, 2004;MarinVicente et al, 2005), and an electron paramagnetic resonance study has demonstrated that this ␤3-4 hairpin lies in close proximity to the membrane surface (Kohout et al, 2003). To investigate the contribution of the ␤3-4 hairpin to specific Figure 5.…”

“…Earlier studies have suggested that lysines 197 and 199 in the ␤3 strand and lysines 209 and 211 in the ␤4 strand of PKC␣C2 are important in Ca 2ϩ -independent, PIP 2 -dependent binding to vesicles, PSand PIP 2 -dependent regulation of PKC␣ activity in vitro, and membrane residence (Ochoa et al, 2002;Corbalan-Garcia et al, 2003;Rodriguez-Alfaro et al, 2004;Marin-Vicente et al, 2005). The hypothesis that these lysine residues are important in PIP 2 recognition predicts that mutations at these positions would weaken membrane-targeting specificity in vivo and lipid-binding affinity in vitro.…”

“…Further structural studies of PKC␣C2 complexed with Ca 2ϩ and soluble PS have revealed a basic region comprised of four lysine residues in the ␤3-4 hairpin that endow the region with a strong positive charge (Ochoa et al, 2002). Specific interactions between K209 and K211 and the soluble PS have been observed in a crystal structure (Ochoa et al, 2002).…”

“…Further structural studies of PKC␣C2 complexed with Ca 2ϩ and soluble PS have revealed a basic region comprised of four lysine residues in the ␤3-4 hairpin that endow the region with a strong positive charge (Ochoa et al, 2002). Specific interactions between K209 and K211 and the soluble PS have been observed in a crystal structure (Ochoa et al, 2002). Moreover, K209 and K211 have been implicated in studies of PIP 2 binding , whereas both PSand PIP 2 -dependent enzyme activation is reduced by mutation of basic region lysines Rodriguez-Alfaro et al, 2004).…”

“…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).…”