Abstract
(1)Structural and functional study of chloroplast translocon component atToc33 ABSTRACT Arabidopsis Toc33 (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg130) has been implicated in formation of the atToc33 dimer and inter-molecular GTPase activation within the dimer. Here we report the crystal structure at 3.2 Å resolution of an atToc33 mutant, atToc33(R130A), in which Arg130 was mutated to alanine. Both in solution and in crystals, atToc33(R130A) was present in its monomeric form. In contrast, both wild-type atToc33 and another pea Toc GTPase homologue, pea Toc159 (psToc159), were able to form dimers in solution. Dimeric atToc33 and psToc159 had significantly higher GTPase activity than monomeric atToc33, psToc159 and atToc33(R130A). Molecular modeling using the structures of psToc34 and atToc33(R130A) suggests that, in an architectural dimer of atToc33, Arg130 from one monomer interacts with the □-phosphate of GDP and several other amino acids of the other monomer. These results indicate that Arg130 is critical for dimer formation, which is itself important for GTPase activity. Activation of GTPase activity by dimer formation is likely to be a critical regulatory step in protein import into chloroplasts. (2)Crystallographic study of cobra phospholipase A2 complexed with fatty acid ABSTRACT Phospholipase A2 (PLA2) exhibit high catalytic activities on aggregated substrates via interfacial activation. Recent biophysical characterizations of PLA2 bound to a membrane surface have suggested that both cooperative binding of anionic amphiphiles to the interface and conformational changes of the regulatory N-terminal helix are involved in interfacial activation, but their specific role in either facilitating substrate diffusion and/or a conformational change at the catalytic site remains to be clarified. Herein, we present crystal structures of cobra (Naja atra) PLA2 in complex with anionic sulfate amphiphiles bound at the interfacial and/or catalytic site; we also determine its orientation against phospholipid membranes using the FTIR method. The results suggest that PLA2 bindings to phospholipid membrane induce a tilting of the hydrocarbon chain of phospholipids with an uneven depth of penetration of aromatic residues of the enzyme into the surface. The interfacial amphiphiles are also shown to diffuse from the interfacial binding site to the active site via binding-induced conformational changes as evidenced by a time-dependent change in the crystal form. Thus, interfacial activation of cobra PLA2 may involve a structural intermediate of the enzyme with interfacially bound amphiphiles to facilitate the diffusion of substrates. The intermediate is suggested to behave similarly to the pre-micellar aggregate of the enzyme and involve the binding of amphiphile to anionic binding cluster region of cobra PLA2 with the hydrocarbon tail of the lipid interacting directly with the hydrophobic amino acid residues located near the N-terminal and the pore region.