Cuppari Anna, Körschgen Hagen, Fahrenkamp Dirk, Schmitz Carlo, Guevara Tibisay, Karmilin Konstantin, Kuske Michael, Olf Mario, Dietzel Eileen, Yiallouros Irene, de Sanctis Daniele, Goulas Theodoros, Weiskirchen Ralf, Jahnen-Dechent Willi, Floehr Julia, Stoecker Walter, Jovine Luca, Gomis-Rüth F Xavier
Proteolysis Laboratory, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, Helix Building, c/o Baldiri Reixac 15-21, E-08028 Barcelona, Catalonia, Spain.
Institute of Molecular Physiology, Cell and Matrix Biology, Johannes Gutenberg-University Mainz, Johann-Joachim-Becher-Weg 7, D-55128 Mainz, Germany.
IUCrJ. 2019 Feb 28;6(Pt 2):317-330. doi: 10.1107/S2052252519001568. eCollection 2019 Mar 1.
Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallo-peptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked 'CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWVGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel 'raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWVGP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.
哺乳动物胎球蛋白-A和胎球蛋白-B是具有多种功能的丰富血清蛋白。胎球蛋白-B是虾红素家族金属肽酶（MPs）的一种高度选择性和强效抑制剂，该家族在哺乳动物中包括卵母细胞astacin。通过抑制卵母细胞astacin，胎球蛋白-B对雌性生育至关重要。已确定胎球蛋白-B未结合状态以及与典型虾红素结合状态的晶体结构，发现该抑制剂具有串联的胱抑素型模块（CY1和CY2）。它们通过一个带有刚性二硫键连接的“CPDCP主干”的暴露连接子相连，随后是一个几乎没有规则二级结构的C末端区域（CTR）。CPDCP主干和CY2的一个发夹形成一个二分楔形，插入到MP的活性位点裂隙中。这些元件分别占据裂隙的非引发侧和引发侧，但留出特异性口袋，因此抑制剂不会被切割。主干中的天冬氨酸阻断虾红素的催化锌，而CY2发夹通过一个QWVGP基序结合。CY1模块有助于结构完整性，而CTR不参与抑制作用，这已通过一系列突变体和变体研究得到证实。总体而言，这种抑制作用符合一种针对MPs的新型“象鼻抬起”机制，这让人联想到靶向半胱氨酸肽酶的单结构域胱抑素。来自脊椎动物的200多个序列已被注释为胎球蛋白-B，这证明了它的普遍性和生理相关性；因此，从哺乳动物到软骨鱼类都发现了具有保守CPDCP和QWVGP衍生基序的序列。因此，象鼻抬起机制可能普遍适用于胎球蛋白-B直系同源物对虾红素的抑制作用。
