Abstract
The tissue inhibitor of metalloproteinases-1 (TIMP-1) exerts inhibitory activity against matrix metalloproteinases and cytokine-like effects. We previously showed that TIMP-1 reduces neurite outgrowth in mouse cortical neurons and that this cytokine-like effect depends on TIMP-1 endocytosis mediated by the low-density lipoprotein receptor-related protein-1 (LRP-1). To gain insight into the interaction between TIMP-1 and LRP-1, we considered conformational changes that occur when a ligand binds to its receptor. TIMP-1 conformational changes have been studied using biomolecular simulations, and our results provide evidence for a hinge region that is critical for the protein movement between the N- and C-terminal TIMP-1 domains. In silico mutants have been proposed on residues F12 and K47, which are located in the hinge region. Biological analyses of these mutants show that F12A or K47A mutation does not alter MMP inhibitory activity but impairs the effect of TIMP-1 on neurite outgrowth. Interestingly, these mutants bind to LRP-1 but are not endocytosed. We conclude that the intrinsic dynamics of TIMP-1 are not involved in its binding to LRP-1 but rather in the initiation of endocytosis and associated biological effects.
Highlights
The tissue inhibitor of metalloproteinase-1 (TIMP-1) is a natural inhibitor of matrix metalloproteinases (MMP) and several a disintegrin and metalloproteinases (ADAM)[1]
We recently showed that tissue inhibitor of metalloproteinases-1 (TIMP-1) decreased neurite outgrowth in cortical neurons and that this effect was mainly dependent on its endocytosis mediated by the low-density lipoprotein receptor-related protein-1 (LRP-1)[6]
TIMP-1 has been described as a protein whose function could be controlled by its intrinsic dynamics[25]
Summary
Determination of TIMP-1 intrinsic dynamics using molecular modelling. TIMP-1 has been described as a protein whose function could be controlled by its intrinsic dynamics[25]. The graph shows nine regions (green arrow) in which the residues exhibited both high deformation energy and low Cα atomic fluctuation These regions were grouped in an area of the TIMP-1 3D structure that could be linked to a hinge essential for the interdomain displacements (Fig. 2b). Our SPR data demonstrated that the lack of neurite outgrowth inhibition could not be explained by a difference of affinity towards LRP-1 between the mutants and T1-WT To support these unexpected results, we performed a competition assay by co-treating cortical neurons with different T1-WT/T1-F12A or T1-WT/T1-K47A ratios (Fig. 5c). In the absence of structural data regarding one of the two partners, considering the intrinsic dynamics of the proteins could provide additional information for studying ligand/receptor interactions and their associated cellular effects
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