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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.

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N-Acetyl-L-glutamate kinase (NAGK), a member of the amino acid kinase family, catalyzes the second and frequently controlling step of arginine synthesis. The Escherichia coli NAGK crystal structure to 1.5 A resolution reveals a 258-residue subunit homodimer nucleated by a central 16-stranded molecular open beta sheet sandwiched between alpha helices. In each subunit, AMPPNP, as an alphabetagamma-phosphate-Mg2+ complex, binds along the sheet C edge, and N-acetyl-L-glutamate binds near the dyadic axis with its gamma-COO- aligned at short distance from the gamma-phosphoryl, indicating associative phosphoryl transfer assisted by: (1) Mg2+ complexation; (2) the positive charges on Lys8, Lys217, and on two helix dipoles; and (3) by hydrogen bonding with the y-phosphate. The structural resemblance with carbamate kinase and the alignment of the sequences suggest that NAGK is a structural and functional prototype for the amino acid kinase family, which differs from other acylphosphate-making devices represented by phosphoglycerate kinase, acetate kinase, and biotin carboxylase.