Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

Abstract

Thermolysin-like proteases (TLPs), a large group of zinc metalloproteases, are synthesized as inactive precursors. TLPs with a long propeptide (∼200 residues) undergo maturation following autoprocessing through an elusive molecular mechanism. We report the first two crystal structures for the autoprocessed complexes of a typical TLP, MCP-02. In the autoprocessed complex, Ala205 shifts upward by 33 Å from the previously covalently linked residue, His204, indicating that, following autocleavage of the peptide bond between His204 and Ala205, a large conformational change from the zymogen to the autoprocessed complex occurs. The eight N-terminal residues (residues Ala205-Gly212) of the catalytic domain form a new β-strand, nestling into two other β-strands. Simultaneously, the apparent T(m) of the autoprocessed complex increases 20 °C compared to that of the zymogen. The stepwise degradation of the propeptide begins with two sequential cuttings at Ser49-Val50 and Gly57-Leu58, which lead to the disassembly of the propeptide and the formation of mature MCP-02. Our findings give new insights into the molecular mechanism of TLP maturation.

Fig. 1.

Schematic domain diagram and time-course maturation of the MCP-02 precursor. (A) The domain composition of the MCP-02 precursor. The 727-residue-long MCP-02 precursor contains four main parts: a signal peptide, a propeptide (FTP and PepSY), a catalytic domain (NTD and CTD), and two PPC domains. The PPC domain is a kind of C-terminal extension in many TLPs. As no evidence shows that the PPC domain has any relationship to the maturation of TLPs, we truncated the two PPC domains in all of our clones of MCP-02 and focused only on the propeptide. (B) The time course of MCP-02 maturation analyzed by SDS-PAGE. Lane 1 shows the unautoprocessed MCP-02 zymogen with an E346A mutation. Lanes 2–6 display the time-course maturation pathway of MCP-02 at 37 °C. The unautoprocessed MCP-02 zymogen gradually converts to the final mature enzyme with the last nine residues cleaved by autolysis.

Fig. 2.

Overall structure of mature MCP-02 and interface analysis of the MCP-02 autoprocessed complex. (A) The overall structure of mature MCP-02. The light blue, light green, and pink colored regions indicate the NTD, the central α-helix, and the CTD, respectively. The catalytic zinc is shown as a gray ball, and the calcium ion in the CTD is shown as a green ball. The side chains of the conserved residues around zinc are shown. (B) The overall structure of the MCP-02 autoprocessed complex and the surface charge distribution of the propeptide and the catalytic domain. The large C-shaped propeptide surrounds the CTD tightly. Two 90° rotated figures display the interface between the propeptide and the catalytic domain. The interaction is mainly mediated by the charge discrepancy between the propeptide and the catalytic domain. The key residues contributing to this interaction are indicated. (C) The close-up view of the conserved hydrophobic core of the FTP domain. A series of hydrophobic residues surrounding Tyr81 in the RY motif form the hydrophobic core of the FTP domain. (D) The complicated network of hydrogen bonds and salt bridges between the FTP domain and the CTD. (E) The interaction located between the PepSY domain and the NTD. Four residues colored in gray pack into the hydrophobic core of this interaction.

Fig. 3.

Mechanism of inhibition by the propeptide to the catalytic domain. (A) Comparison of the catalytic sites in the mature MCP-02 and the autoprocessed complexes with the E346A mutation. The last residue (His204) of the propeptide replaces the activated water molecule and forms a new ligand to the zinc in the autoprocessed complex. (B) Binding of the propeptide to the catalytic cleft via the main chain. The last two residues of the propeptide form very short antiparallel β-sheets with Tyr319 and Trp320. (C) The relative inhibitory effects of different propeptide mutants on the catalytic domain. The relative inhibitory effect of each propeptide mutant is shown as the percentage of its inhibitory effect to that of the WT propeptide. The inhibitory effect of the WT propeptide is defined as 100%. Pep-T refers to the propeptide with truncation Thr176-Ser182. In this assay, the BSA (1 mg/mL) was used as substrate. The concentration of the protease used was 100 nM, and the concentration of the propeptide and its derivative mutants was 10 μM.

Fig. 4.

Large changes in conformation and thermostability during the conversion from the zymogen to the autoprocessed complex. (A) A top view of the relatively independent motif from the NTD of the catalytic domain. In the autoprocessed complex, Ala205 has a shift of up to 33 Å from the previously covalently linked residue, His204. The eight N-terminal residues of the catalytic domain form a new β-strand, inserting itself into two other β-strands. (B) A close-up view of the simulated structure of the region involved in the conformational shift from the zymogen model. (C) Thermodenaturation curves of the unautoprocessed zymogen (red line) and the autoprocessed complex (blue line).

Fig. 5.

Stepwise degradation of the propeptide in the MCP-02 autoprocessed complex. (A) The α-helix (red) between Val50 and Leu58. The short α-helix (red) between Val50 and Leu58 is part of the hydrophobic center of the FTP domain, and this α-helix will be released after the sequential cleavages at Ser49-Val50 and Gly57-Leu58. (B) Block of the propeptide degradation by the Val50 and Leu58 to Pro mutations. The Val50 and Leu58 to Pro mutations can block the propeptide degradation to a large extent.

Fig. 6.

The schematic diagrams of the autoprocessing pathway of the zinc metalloprotease MCP-02. From newly synthesized polypeptide to unautoprocessed zymogen, the propeptide binds to the catalytic domain with both of the FTP and PepSY domains. After the first autocleavage, the C terminus of the propeptide alters its binding mode and becomes an inhibitor to the catalytic domain, and then an energy-driven transition occurs, releasing the N terminus of catalytic domain from the catalytic cleft. The degradation of the propeptide starts with cleavages at site 1 and site 2, which are located at the two ends of a short α-helix. These two cuttings result in disassembly of the FTP domain and release of the propeptide from the catalytic domain, which leads to the activation of the protease. The red dot represents the catalytic center of MCP-02.