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Boundary of the nucleotide-binding domain of Streptococcus ComA based on functional and structural analysis.

Biochemistry (2013-03-29)
Seiji Ishii, Takato Yano, Akihiro Okamoto, Takeshi Murakawa, Hideyuki Hayashi
ABSTRACT

The ATP-binding cassette (ABC) transporter ComA is a key molecule essential for the first step of the quorum-sensing system of Streptococcus. The nucleotide binding domains (NBD) of Streptococcus mutans ComA with different N termini, NBD1 (amino acid residues 495-760), NBD2 (517-760), and NBD3 (528-760), were expressed, purified, and characterized. The shortest NBD3 corresponds to the region commonly defined as NBD in the database searches of ABC transporters. A kinetic analysis showed that the extra N-terminal region conferred a significantly higher ATP hydrolytic activity on the NBD at a neutral pH. Gel-filtration, X-ray crystallography, and mutational analyses suggest that at least four to five residues beyond the N-terminal boundary of NBD3 indeed participate in stabilizing the protein scaffold of the domain structure, thereby facilitating the ATP-dependent dimerization of NBD which is a prerequisite to the catalysis. These findings, together with the presence of a highly conserved glycine residue in this region, support the redefinition of the N-terminal boundary of the NBD of these types of ABC exporters.

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