Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition

Abstract

Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallopeptidases (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 QWVXGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro 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 QWVXGP-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.

Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis

“…In contrast to hydroxyapatite crystals, there were also additional peaks at 1450 and 1670 cm −1 originated from the presence of proteins in the particles. The detected β-sheet amide I peak at 1670 cm −1 was expected for fetuin proteins 37 .…”

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases

“…Specific inhibition of tolloid astacins has been reported for Xenopus laevis sizzled/ogon 31,32 . By contrast, meprins, crayfish astacin, nephrosin from cyprinid fishes, and ovastacin are strongly inhibited by fetuin-B forms from mammals, which are strictly selective for astacins 33–36 , and by fish fetuin, which acts as the physiological antagonist of nephrosin 37 . By blocking ovastacin, fetuin-B prevents premature hardening of the zona pellucida and maintains female fertility 26,33,34 .…”

“…Within the family, fetuins are type-3 cystatins (subfamily I25C), which include glycosylated proteins with two or three cystatin-like modules 40,41 . Recent crystal structures of the mouse ortholog (mFB), isolated and in complex with crayfish astacin 36 , have revealed that the inhibitor consists of the tandem cystatin-type modules 1 and 2 (CY1 and CY2), which are united by a linker (LNK) with a “CPDCP-trunk” and followed by a C-terminal region (CTR). The inhibitor blocks the active-site cleft of the MP following a novel “raised-elephant-trunk” mechanism 36 .…”

Mammalian egg coat modifications and the block to polyspermy

“…Under these circumstances, serum glycoprotein fetuin‐B, a cystatin superfamily metalloprotease inhibitor that is essential for mouse fertility (Dietzel et al, 2013), was shown to maintain in vitro fertilization by counteracting ovastacin‐mediated cleavage of ZP2 (Dietzel, Floehr, van de Leur, Weiskirchen, & Jahnen‐Dechent, 2017; Dietzel et al, 2013; Floehr et al, 2016). A recent crystallographic study of fetuin‐B and its complex with crayfish astacin, a nonphysiological binding partner that resembles ovastacin, suggested that the function of the inhibitor depends on a rigid disulfide‐linked CPDCP motif located between its two cystatin‐type modules (Cuppari et al, 2019; Figure 2).…”

“…mediated cleavage of ZP2(Dietzel, Floehr, van de Leur, Weiskirchen, & Jahnen-Dechent, 2017;Dietzel et al, 2013;Floehr et al, 2016). A recent crystallographic study of fetuin-B and its complex with crayfish astacin, a nonphysiological binding partner that resembles ovastacin, suggested that the function of the inhibitor depends on a rigid disulfide-linked CPDCP motif located between its two cystatin-type modules(Cuppari et al, 2019; Figure 2). Activated oocytes and two-cell embryos do not show complete ZP2 cleavage.…”