Novel Insights into the Catalytic Mechanism of Collagenolysis by Zn(II)-Dependent Matrix Metalloproteinase-1

Abstract

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.

Figure 1

(a) Matrix metalloproteinase-1 bound to THP and (b) the QM-part considered for calculations.

Scheme 1. Schematic Representation of the Catalytic Steps Explored in This Study

The reaction progresses through A–H. From A1 to B, a proton transfers to Glu219 via an auxiliary water molecule. In step 4a, the proton transfers from the auxiliary water molecule before the C–N bond cleavage.

Figure 2

(a) CVs and (b) FES for step 1. Structures corresponding to reactant (A), TS (TS1), and product (B). Red dotted line indicates the hydrogen bonds.

Figure 3

(a) CVs and (b) FES generated for step 2. Snapshots of structures correspond to reactant (B), TSs (TS2 and TS3), intermediate (IM1), and product (C).

Figure 4

(a) CVs and (b) FES generated for step 3. Snapshots of structures corresponding to reactant (C), product (D), and TS (TS4) hydrogen bond rearrangement stationary points were given. Red dotted lines represent the hydrogen bonds.

Figure 5

(a) CVs and (b) FES generated for step 4a. Snapshots of structures corresponding to reactant (D), product (E), and TS (TS5) of this step. Red color lines represent the hydrogen bonds.

Figure 6

(a) CVs and (b) FES generated for step 4b. Snapshots of structures correspond to reactant (D), IM2, product (F), and TS (TS6 and TS7). Hydrogen bond between water molecules and N-terminals shown in blue color.

Figure 7

(a) CVs and (b) FES generated for step 5. Snapshots of structures correspond to reactant (E), IM3, product (F), and TS (TS8 and TS9).

Figure 8

(a) CVs and (b) FES generated for step 6. Snapshots of structures correspond to reactant (F), IM4, product (G), and TS (TS10 and TS11) of the C–N cleavage step.

Figure 9

(a) CVs and (b) FES generated for step 7. Snapshots of structures corresponding to reactant (G), product (H) and TS (TS12).

Figure 10

Free energy profile through the MetD simulations as per Scheme 1 in the case of complete protein QM/MM in explicit water molecules. Dotted lines, A to B is for the proton transfer from the auxiliary water molecule, while D to F is for the step 4a and step 5 of Scheme 1. All the data in black color are reported w.r.t initial reactant A. Activation barriers w.r.t immediate IM are given in red color.