Akiba Toshihiko, Abe Yuichi, Kitada Sakae, Kusaka Yoshitomo, Ito Akio, Ichimatsu Tokio, Katayama Hideki, Akao Tetsuyuki, Higuchi Kazuhiko, Mizuki Eiichi, Ohba Michio, Kanai Ryuta, Harata Kazuaki
Biological Information Research Center, AIST, Tsukuba, Ibaraki 305-8566, Japan.
J Mol Biol. 2009 Feb 13;386(1):121-33. doi: 10.1016/j.jmb.2008.12.002. Epub 2008 Dec 9.
Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.
副孢子素-2是一种蛋白质毒素，从革兰氏阳性细菌苏云金芽孢杆菌的伴孢晶体中分离得到。尽管苏云金芽孢杆菌通常被认为是杀虫毒素的宝贵来源，但副孢子素-2并不具有杀虫活性，而是对肝癌细胞和结肠癌细胞具有很强的杀细胞活性。37 kDa的无活性新生蛋白经蛋白水解切割成30 kDa的活性形式，该活性形式失去了N端和C端片段。对副孢子素-2积累的细胞学和生化观察表明，该蛋白是一种成孔毒素。为了证实这一假设，我们以2.38 Å的分辨率确定了其活性形式的晶体结构。该蛋白异常细长，主要由与其长轴对齐的长β链组成。它类似于气单胞菌溶素型β-成孔毒素，这有力地支持了成孔假设。该分子可分为三个结构域。结构域1由一个被短α螺旋夹在中间的小β折叠组成，可能是靶标结合模块。另外两个结构域都是β折叠片，被认为与寡聚化和成孔有关。结构域2具有气单胞菌溶素型毒素特有的假定通道形成β发夹结构。该蛋白表面有一条由丝氨酸和苏氨酸残基暴露侧链组成的广泛轨迹。当结构域1与靶标结合时，这条轨迹可能使分子定位于细胞膜上，直到启动寡聚化和成孔过程。β发夹结构非常紧密，似乎不太可能像最近为气单胞菌溶素型毒素开发的成孔模型所假设的那样重新形成。提出了一种安全锁模型作为N端抑制片段的失活机制。
