Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping. (14th January 2015)

• Record Type: Journal Article
• Authors: Yamanaka, Masaru; Nagao, Satoshi; Komori, Hirofumi; Higuchi, Yoshiki; Hirota, Shun
• Is Part Of: Protein science. Volume 24:Number 3(2015:Mar.)
• Page Start: 366
• Page End: 375
• Publication Date: 2015-01-14
• Subjects: Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.2627
• Languages: English
• ISSNs: 0961-8368

Abstract

<abstract abstract-type="main"> <title>ABSTRACT</title> <p>Cytochrome <italic>c</italic><sub>555</sub> from hyperthermophilic bacteria <italic>Aquifex aeolicus</italic> (AA cyt <italic>c</italic><sub>555</sub>) is a hyperstable protein belonging to the cyt <italic>c</italic> protein family, which possesses a unique long 3<sub>10</sub>‐α‐3<sub>10</sub> helix containing the heme‐ligating Met61. Herein, we show that AA cyt <italic>c</italic><sub>555</sub> forms dimers by swapping the region containing the extra 3<sub>10</sub>‐α‐3<sub>10</sub> helix and C‐terminal α‐helix. The asymmetric unit of the crystal of dimeric AA cyt <italic>c</italic><sub>555</sub> contained two dimer structures, where the structure of the hinge region (Val53–Lys57) was different among all four protomers. Dimeric AA cyt <italic>c</italic><sub>555</sub> dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt <italic>c</italic><sub>555</sub> were maintained at pH 2.2–11.0. CN<sup>‐</sup> and CO bound to dimeric AA cyt <italic>c</italic><sub>555</sub> in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN<sup>‐</sup> and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2–11.0), respectively, in a wide range of temperature (25–85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt <italic>c</italic><sub>555</sub> changes upon dimerization by domain swapping.</p> </abstract>