Abstract
Procollagen C-proteinase enhancer (PCOLCE) proteins are extracellular matrix proteins that enhance the activities of procollagen C-proteinases by binding to the C-propeptide of procollagen I. PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain. While the enhancement of proteinase activity can be ascribed solely to the CUB domains, sequence homology of the NTR domain with tissue inhibitors of metalloproteinases suggest proteinase inhibitory activity for the NTR domain. Here we present the three-dimensional structure of the NTR domain of human PCOLCE1 as the first example of a structural domain with the canonical features of an NTR module. The structure rules out a binding mode to metalloproteinases similar to that of tissue inhibitors of metalloproteinases but suggests possible inhibitory function toward specific serine proteinases. Sequence conservation between 13 PCOLCE proteins from different organisms suggests a conserved binding surface for other protein partners.
Highlights
PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain
Human PCOLCE1 is a 50-kDa protein (55 kDa when glycosylated) [2] of a rod-like shape of about 150 Å length [5]. It is built of three structural modules, comprising two CUB domains followed by a C-terminal netrin-like (NTR) domain
The enhancement in bone morphogenetic protein-1 (BMP-1) activity by PCOLCE1 has been assigned to the CUB domains, as the enhancer activity persists after removal of the C-terminal NTR domain by natural processing [2, 3]
Summary
In the case of human plasma kallikrein the enzyme (3 nM) was preincubated for 30 min at 37 °C in 50 mM Tris, 100 mM NaCl, 2 mM CaCl2, 0.01% Triton X-100, pH 7.5 buffer, in the presence of increasing concentrations of thrombin-digested recombinant NTRPCOLCE1 (10 – 60 M), and the reactions were initiated by adding the substrate D-Pro-Phe-Arg-pNA at 650 M final concentration. Structural conservation between the NTR domains from human PCOLCE1 and other PCOLCE NTR domains is indicated by about 35% sequence identity [6, 28] and by the fact that the buried side chains in NTRPCOLCE1 (Fig. 1B) are almost always hydrophobic or uncharged in the other PCOLCE proteins (Fig. 1B).
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