The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases
The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases
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DOI：
10.1038/s41598-019-51095-y
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发表时间：
2019-10
影响因子：
4.6
通讯作者：
T. Guevara;Hagen Körschgen;A. Cuppari;C. Schmitz;Michael Kuske;I. Yiallouros;J. Floehr;W. Jahnen-Dechent;W. Stöcker;F. Gomis-Rüth
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作者：
T. Guevara;Hagen Körschgen;A. Cuppari;C. Schmitz;Michael Kuske;I. Yiallouros;J. Floehr;W. Jahnen-Dechent;W. Stöcker;F. Gomis-Rüth
人胎球蛋白-B通过其对金属肽酶的astacin家族的成员ovastacin的抑制作用在人类生育中起关键的生理作用。该抑制剂由串联的半胱氨酸蛋白酶抑制剂样结构域（CY 1和CY 2）组成，它们通过含有“CPDCP-主干”的接头连接，然后是没有规则二级结构的C-末端区域（CTR）。在这里，我们解决了抑制剂的复合物的晶体结构的原型虾，这是一个有用的模型，人类ovastacin。来自CY 2的两个发夹，接头和CY 1的“豆荚蛋白结合环”的尖端，根据最近报道的小鼠胎球蛋白B的“升高的象鼻机制”抑制小龙虾虾红素。这种抑制作用是通过阻断催化锌离子上游和下游的活性位点裂缝子位点而发挥的，但不是那些侧翼的易裂键。然而，与小鼠复合物相反，小鼠复合物是用胎球蛋白-B在单个位点切口但在其他方面完整获得的，大部分CTR在人复合物的结晶过程中被蛋白水解除去。此外，晶体学不对称单元中存在的两种复合物在CY 1和CY 2的相对排列中发散，而小鼠复合物晶体结构中发现的两种复合物是等效的。体外生物化学研究证实了人和小鼠胎球蛋白-B在小龙虾虾红素前的差异切割敏感性，并显示切割的人抑制剂阻断小龙虾虾红素和人美普林α和β的效力仅略低于完整变体。因此，动物胎球蛋白-B直系同源物的CTR可能具有维持CY 1和CY 2的特定相对取向的功能，尽管如此，该相对取向对于肽酶抑制是不利的。
Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studiesin vitroconfirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.