Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.

Midkine (MK) is a 13 kDa heparin‐binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C‐terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel β‐strands, but the C‐terminal domain has a long flexible hairpin loop where a heparin‐binding consensus sequence is located. Basic residues on the β‐sheet of the C‐terminal domain form another heparin‐binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head‐to‐head dimer model of MK. The model was supported by the results of cross‐linking experiments using transglutaminase. The dimer has a fused heparin‐binding site at the dimer interface of the C‐terminal domain, and the heparin‐binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin‐binding activity and biological activity of the dimer.

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-Å resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V₁ (soluble) and the V_o (membrane bound) domains. Subunit C, attached to the V_o domain, seems to have a socket like function in attaching the central-stalk subunits of the V₁ domain. This architecture seems essential for the reversible association/dissociation of the V₁ and the V_o domains, unique for V-ATPase activity regulation.

Vav is a guanine nucleotide exchange factor for the Rho/Rac family that is expressed exclusively in hematopoietic cells. Growth factor receptor‐bound protein 2 (Grb2) has been proposed to play important roles in the membrane localization and activation of Vav through dimerization of its C‐terminal Src‐homology 3 (SH3) domain (GrbS) and the N‐terminal SH3 domain of Vav (VavS). The crystal structure of VavS complexed with GrbS has been solved. VavS is distinct from other SH3 domain proteins in that its binding site for proline‐rich peptides is blocked by its own RT loop. One of the ends of the VavS β‐barrel forms a concave hydrophobic surface. The GrbS components make a contiguous complementary interface with the VavS surface. The binding site of GrbS for VavS partially overlaps with the canonical binding site for proline‐rich peptides, but is definitely different. Mutations at the interface caused a decrease in the binding affinity of VavS for GrbS by 4‐ to 40‐fold. The structure reveals how GrbS discriminates VavS specifically from other signaling molecules without binding to the proline‐rich motif.