Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer

Rangita Dawadi,Jan-Olof Winberg,Nabin Malla,Imin Wushur,Eli Berg,Ingebrigt Sylte,Beate Hegge,Gunbjørg Svineng

doi:10.3390/ijms21124205

Rangita Dawadi, Jan-Olof Winberg + Show 6 more

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https://doi.org/10.3390/ijms21124205

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Abstract

Previous studies have shown that THP-1 cells produced an SDS-stable and reduction-sensitive complex between proMMP-9 and a chondroitin sulfate proteoglycan (CSPG) core protein. The complex could be reconstituted in vitro using purified serglycin (SG) and proMMP-9 and contained no inter-disulfide bridges. It was suggested that the complex involved both the FnII module and HPX domain of proMMP-9. The aims of the present study were to resolve the interacting regions of the molecules that form the complex and the types of interactions involved. In order to study this, we expressed and purified full-length and deletion variants of proMMP-9, purified CSPG and SG, and performed in vitro reconstitution assays, peptide arrays, protein modelling, docking, and molecular dynamics (MD) simulations. ProMMP-9 variants lacking both the FnII module and the HPX domain did not form the proMMP-9∙CSPG/SG complex. Deletion variants containing at least the FnII module or the HPX domain formed the proMMP-9∙CSPG/SG complex, as did the SG core protein without CS chains. The interacting parts covered large surface areas of both molecules and implicated dynamic and complementary ionic, hydrophobic, and hydrogen bond interactions. Hence, no short single interacting linear motifs in the two macromolecules could explain the strong SDS-stable and reduction-sensitive binding.

Highlights

The matrix metalloprotease (MMP) or matrixin family is a group of extracellular zinc and calcium dependent metallo-enzymes expressed by most cells and tissues
The purified and partly purified proMMP-9 variants were subjected to gelatin zymography (Figure 1B), sodium dodecyl sulfate (SDS)-PAGE, and Western blotting (Figure 2)
Crude media containing proMMP-9 deletion variants were subjected to real-time gelatin zymography (Figure 1C) and Western blotting (Figure 2)

Summary

Introduction

The matrix metalloprotease (MMP) or matrixin family is a group of extracellular zinc and calcium dependent metallo-enzymes expressed by most cells and tissues. The MMPs as a group, as well as individual enzymes, have broad substrate specificity, and together, they are able to degrade almost all extracellular matrix proteins They cleave a number of non-extracellular matrix molecules, such as cytokines, chemokines, adhesion molecules, cell receptors, proteases (including MMPs), protease inhibitors, and a number of intracellular proteins [1,2,3,4,5,6,7,8]. Due to their broad substrate specificity, MMPs play a critical role in cell and tissue homeostasis and normal development, while dysregulation during disease may be either detrimental or protective to the organism. The numerous interactions between proteins and other molecules regulate their accessibility to proteolytic degradation, by either hiding protease cleavage sites or exposing new sites

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