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.zymogen conversion | zinc metalloproteases inhibition | intramolecular chaperone T he thermolysin (M4) family is comprised of a large number of zinc metalloproteases in the subclan MA(E), most of which have similar sequences and domain structures (1). The representative member of this family is thermolysin (EC 3.4.24.27), a 34.6-kDa thermostable metalloprotease secreted by Bacillus thermoproteolyticus (2). The amino acid sequence and the threedimensional structure of thermolysin were first determined in 1972 (3, 4). Since then, thermolysin and thermolysin-like proteases (TLPs) have been used as models in studying zinc metalloproteases (5, 6).TLPs are widely distributed in many species of microorganisms, and many TLPs are regarded as key pathogenesis factors that are responsible for some severe bacterial infections (7). For example, λ-toxin from Clostridium perfringens can degrade various human immune defense proteins (8). Vibriolysin from Vibrio vulnificus (9) and pseudolysin from Pseudomonas aeruginosa (10) can be lethally blood-poisonous to humans. Therefore, understanding the catalytic and maturation mechanisms of TLPs is very important for developing therapeutics to treat such infectious diseases.TLPs are synthesized as a precursor with a propeptide. TLPs are divided into two distinct groups according to the primary structure of their propeptides. Less than 30% of the total TLPs have short propeptides (∼50 residues), and more than 70% of TLPs have long propeptides (∼200 residues) (11, 12). The long propeptide in TLPs contains two distinguished regions of conservation: an FTP (fungalysin/thermolysin propeptide) domain and a PepSY [peptidase propeptide and YPEB (a protein encoded by Bacillus subtilis ypeB gene)] domain. To date, the PepSY domain has been found not only in TLPs, but also in many nonpeptidase proteins, wherein the presence of this domain may prevent detrimental protease ...

A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases

“…By contrast, the insertion of the C-terminal tail into the active-site cleft in the reverse orientation of a substrate, thus blocking structural elements essential for catalysis and access of true substrates, provides a structural explanation for the lack of hydrolytic activity of the protein as it would actually correspond to a zymogen. The latency mode has been previously unseen for structurally characterized MPs, which are normally kept latent by N-terminal pro-domains or propeptides, as in astacin (80), meprin ␤ (81), fragilysin (52), funnelin carboxypeptidases (82), matrix metalloproteinases (77), ADAMs/adamalysins (78), and thermolysin (83). Accordingly, the present structure represents a novel mechanism of intramolecular latency maintenance in MPs.…”

“…After ␣2, the polypeptide chain enters the C-terminal subdomain through L␣2␣3, which leads to the "glutamate helix" ␣3. The latter is termed thus due to its topological equivalence with a similar helix in gluzincins (see below), and it lies in a horizontal plane that is roughly parallel to that of helix ␣2 but with the helix axis vertically rotated backwards by ϳ50°, so that ␣3 ends with V 83 at the back molecular surface. At F 84 , the chain turns frontward and, after a loop that forms the bottom of the molecule (P 85 -R 86 ), opens out into the "C-terminal helix" ␣4, which runs obliquely and creates the lower right part of the molecule (Fig.…”

The Metalloprotease of Listeria monocytogenes Is Regulated by pH

“…Degradation of the propeptide by its cognate catalytic domain is essential for full activation of the protease (8). We would argue that this mechanism of regulation does not apply to Mpl for two reasons.…”

Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores

“…In order to predict residues of the YpeB PepSY domains that might be important for protein-protein interactions, the PepSY domain sequences from B. anthracis YpeB were aligned with a PepSY domain consensus sequence (45), other PepSY domain sequences (40,45,46), and the YpeB PepSY domain sequences from other Bacillus species by using Clustal W sequence alignment software (47). Based on these alignments, highly conserved residues were identified as targets for mutagenesis.…”