Akeo Shinkai

Mapping of the Sites Involved in Ligand Association and Dissociation at the Extracellular Domain of the Kinase Insert Domain-containing Receptor for Vascular Endothelial Growth Factor

The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.

SecA, an essential component of the secretory machinery of Escherichiacoli, exists as homodimer

Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of Escherichia coli suggested that SecA, an essential component of the secretory machinery, exists as an oligomer. The subunit structure of SecA was then studied using a purified specimen. Estimation of the molecular mass by means of ultracentrifugation and chemical crosslinking analysis revealed that SecA exists as a homodimer. The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol. It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.

Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in Escherichiacoli

SecA is an essential component of the protein secretory machinery of Escherichia coli. SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis. The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structures. It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation. SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution. Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol. It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.