Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Abstract

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.

Fig. 1.

Structure of promeprin β. (A) The structure of the pMβΔC monomer shown in front (Left) (CD in standard orientation according to ref. 41) and top (Right) reference views. The latter is along the sugar channel (green arrow). Glycans are depicted as stick models and the respective asparagine residues are numbered. The corresponding surface models are depicted above each picture (glycans in white). PD is shown in ochre, CD in aquamarine, MAM in red, and TRAF in purple. The zinc and the sodium ions are shown as magenta and blue spheres, respectively. (B) Close-up view in stereo of A, Left, to highlight residues engaged in PD–CD interactions. (C) Same as B in mono for the interaction between PD and TRAF. The first two residues of the structure (P²³–W²⁴) are actually T²³–P²⁴ in the natural protein (SI Materials and Methods). (D) pMβΔC dimer superposed with its Connolly surface shown in the front (Left) (in a plane with the membrane) and bottom (Right) dimer reference views. PDs are shown in ochre and yellow, CDs in aquamarine and blue, MAMs in red and green, TRAFs in purple and pink, and sugar moieties in white and gray. The intermolecular twofold axis is shown in red and green arrows point at the sugar channels. (E) Cartoon depicting the dimer as a ribbon in front (Left) and bottom (Right) views as in D. Green arrows run along the sugar channels and a pink arrow highlights the cleft of one CD with the adequate orientation of a substrate. The segment containing the intermolecular disulfide bond between C³⁰⁵ residues is disordered in the zymogen and its approximate position is highlighted by orange ellipses.

Fig. 2.

Sheddase mechanism of meprin β. Shown is a working model of Mβ function based on the experimental dimer (CDs in aquamarine and turquoise, MAMs in orange and red, TRAFs in mauve and purple, and glycosylation moieties in light green) in front (Upper) view (Fig. 1D, Left) and top (from the membrane surface; Lower) view from the membrane (here the membrane was removed for clarity). The segments present in the Mβ dimer but missing in the experimental MβΔC structure [EGF, transmembrane (TM), and cytosolic tail (CST)] have been computationally modeled (SI Materials and Methods) and are shown in white/light blue. A transmembrane substrate model for APP (segment 624–723) is shown as a blue ribbon. The substrate segment proposed to interact with the dimer partner is depicted in pale blue to highlight that this part of the mechanism is more speculative. APP glycosylation sites relevant for the proposed mechanism are marked in yellow on the ribbon and labeled. (Upper) Red ellipses highlight sugars attached to N⁴³⁶ of the right monomer (lower ellipse) and to N²⁵⁴ of both monomers (upper ellipse). (Lower) Image shows only the latter ellipse. (Upper Right Inset) A possible “N-like” trajectory of the substrate.