The roles of protein conformational dynamics and allostery in function are well-known. However, the roles that interdomain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between interdomain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the interdomain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using (1) an active site mutant (E219Q) of MMP1, (2) MMP9, another member of the MMP family that increases the activity of MMP1, and (3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent interdomain dynamics may enable allosteric control of the MMP1 function.

AbstractThe roles of protein conformational dynamics and allostery in function are well-known. However, the roles that inter-domain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between inter-domain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the inter-domain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using 1) an active site mutant (E219Q) of MMP1, 2) MMP9, another member of the MMP family that increases the activity of MMP1, and 3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent inter-domain dynamics may enable allosteric control of the MMP1 function.

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Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Amyloid-beta peptide (Aβ) is the primary component of water-insoluble extracellular plaques, one of the critical hallmarks of Alzheimer's disease (AD). Matrix metalloproteases (MMPs) are broad-spectrum proteases with diverse functions, including interactions with Aβ. Here we report single molecule measurements of MMP1 dynamics on Aβ-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains. We show that the two domains of MMP1 prefer closed conformations on Aβ-induced aggregates, in contrast to the preference for open conformations on collagen fibril, fibrin, and alpha-synuclein aggregates. We approximated the MMP1 dynamics by a two-state Poisson process and determined the kinetic rates of interconversion between the two states from histograms and correlations of FRET values. We performed molecular docking of MMP1 with Aβ using ClusPro, simulated MMP1 dynamics using different docking poses, and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used simulations to create a two-dimensional map of correlations between every pair of MMP1 residues, which shows allosteric communications between the two MMP1 domains. We calculated a Gray Level Co-occurrence Matrix from the two-dimensional map of correlations and quantified MMP1 fluctuations by Shannon entropy. We identified the allosteric residues in the hemopexin domain by identifying residues having strong correlations with the catalytic motif residues. We identified that the residues I364, G369, P409, G410, and D418 in MMP1 have Aβ-specific allosteric correlations with the MMP1 catalytic motif by comparing residues for free and Aβ-bound MMP1. We used these Aβ-specific allosteric residues to select small molecule ligands after the virtual screening of molecules against Aβ-bound MMP1. Molecular understanding of interactions between MMP1 and Aβ-induced aggregates and identification of substrate-specific allosteric residues may enable controlling MMP1 function selectively on Aβ.