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

Anna Cuppari,Luca Jovine,Irene Yiallouros,Mario Olf,Daniele De Sanctis,Willi Jahnen-Dechent,F Xavier Gomis-Rüth,Hagen Körschgen,Carlo Schmitz,Tibisay Guevara,Dirk Fahrenkamp,Ralf Weiskirchen,Michael Kuske,Theodoros Goulas,Konstantin Karmilin,Julia Floehr,Eileen Dietzel,Walter Stoecker

doi:10.1107/s2052252519001568

Abstract

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 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.

Highlights

Fetuin was discovered in 1944 as the most abundant globulin in fetal calf serum (Pedersen, 1944)
Examination of the crystal packing of the protein expressed in mammalian cells explains why it crystallized in its unbound form, whereas the material produced in insect cells did not: endoglycosidase H processing of the high-mannose N-glycans of the HEK293Sderived protein left a single N-acetylglucosamine moiety attached to N40 that stacks against W200 of a symmetry-related molecule (Supplementary Fig. S1)
This introduces a crucial crystal-packing contact that would most likely be hindered by the presence of additional sugar residues attached to the core N-acetylglucosamine, such as those normally found in proteins expressed in insect cells

Summary

Introduction

Fetuin was discovered in 1944 as the most abundant globulin in fetal calf serum (Pedersen, 1944). This family is derived from the archetypal reversible inhibitor specific for cysteine peptidases, the monomeric 116-residue chicken egg-white cystatin (ovocystatin; Bode et al, 1988; Stubbs et al, 1990). Fetuins consist of tandem cystatin domains (hereafter referred to as CY1 and CY2) followed by a C-terminal region (hereafter referred to as CTR) Another member of this subfamily is histidine-rich glycoprotein, a tworepeat plasma protein from vertebrates that is involved in the vascular, coagulation and immune systems and is not a peptidase inhibitor (Turk & Bode, 1991). These inhibitors contain two cystatin modules and operate through an unknown mechanism

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Conclusion