Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp SM9913 BINDING OF THE PKD DOMAIN TO COLLAGEN RESULTS IN COLLAGEN SWELLING BUT DOES NOT UNWIND THE COLLAGEN TRIPLE HELIX
Mechanistic Insight into the Function of the C-terminal PKD Domain of the Collagenolytic Serine Protease Deseasin MCP-01 from Deep Sea Pseudoalteromonas sp SM9913 BINDING OF THE PKD DOMAIN TO COLLAGEN RESULTS IN COLLAGEN SWELLING BUT DOES NOT UNWIND THE COLLAGEN TRIPLE HELIX
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DOI：
10.1074/jbc.m109.087023
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发表时间：
2010-05-07
影响因子：
4.8
通讯作者：
Zhang, Yu-Zhong
中科院分区：
文献类型：
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作者：
Wang, Yu-Kai;Zhao, Guo-Yan;Zhang, Yu-Zhong
Deseasin MCP-01是一种细菌型胶原蛋白丝氨酸蛋白酶。其催化结构域可单独降解胶原，其C端的PKD结构域是一个胶原结合结构域(CBD)，可通过未知的机制提高催化结构域的胶原酶分解效率。采用扫描电子显微镜(SEM)、原子力显微镜(AFM)、Zeta电位和圆二色谱研究了PKD结构域在MCP-01胶原酶降解中的作用机制。PKD结构域明显肿胀不溶性胶原。其对胶原蛋白的溶胀能力和对催化区胶原酶活性的改善均依赖于温度。扫描电子显微镜观察显示，处理1h后，PKD结构域胶原束肿胀，直径由5.3微米增大至8.8微米，形成胶原束的纤维分散。AFM观察直接显示PKD结构域与胶原结合，微纤维肿胀，单体暴露。PKD突变体W36A既不结合胶原，也不干扰其结构。Zeta电位结果表明，PKD处理增加了胶原表面的净正电荷。PKD处理对胶原三螺旋的含量和热稳定性没有影响。此外，经PKD处理的胶原蛋白不能被明胶酶降解。因此，虽然暴露了三螺旋单体，但PKD结构域不能解离胶原三螺旋。我们的研究揭示了胶原酶MCP-01的PKD结构域在胶原降解中的作用机制，这与哺乳动物基质金属蛋白酶的CBD结构域不同。
Deseasin MCP-01 is a bacterial collagenolytic serine protease. Its catalytic domain alone can degrade collagen, and its C-terminal PKD domain is a collagen-binding domain (CBD) that can improve the collagenolytic efficiency of the catalytic domain by an unknown mechanism. Here, scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, and circular dichroism spectroscopy were used to clarify the functional mechanism of the PKD domain in MCP-01 collagenolysis. The PKD domain observably swelled insoluble collagen. Its collagen-swelling ability and its improvement to the collagenolysis of the catalytic domain are both temperature-dependent. SEM observation showed the PKD domain swelled collagen fascicles with an increase of their diameter from 5.3 mu m to 8.8 mu m after 1 h of treatment, and the fibrils forming the fascicles were dispersed. AFM observation directly showed that the PKD domain bound collagen, swelled the microfibrils, and exposed the monomers. The PKD mutant W36A neither bound collagen nor disturbed its structure. Zeta potential results demonstrated that PKD treatment increased the net positive charges of the collagen surface. PKD treatment caused no change in the content or the thermostability of the collagen triple helix. Furthermore, the PKD-treated collagen could not be degraded by gelatinase. Therefore, though the triple helix monomers were exposed, the PKD domain could not unwind the collagen triple helix. Our study reveals the functional mechanism of the PKD domain of the collagenolytic serine protease MCP-01 in collagen degradation, which is distinct from that of the CBDs of mammalian matrix metalloproteases.