Structural insights into thrombolytic activity of destabilase from medicinal leech

Abstract

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

Comparative genomics of three non-hematophagous leeches (Whitmaniaspp.): focusing on antithrombotic genes

“…The reactive residues lysine and isoleucine (PKILK) of the archetypal LDTI (Sommerhoff et al 1994) were conserved in the LDTIs of W. laevis and W. pigra, but not those of W. acranulata (Figure S9). The catalytic histidine (KIHNM) of the archetypal destabilase (Marin et al 2023) was conserved in all destabilases from the three Whitmania species, except for destabilase_Wlae5 (Figure S14).…”

A Chromosome-Level Genome Assembly of the Non-Hematophagous Leech Whitmania pigra (Whitman 1884): Identification and Expression Analysis of Antithrombotic Genes

“…The destabilases and GGTs, but not the LCI, had a signal peptide. Moreover, the catalytic residues, histidine of destabilases [52], and threonine of GGTs [53] from both the W. pigra and H. manillensis were conserved. In total, four destabilase genes were detected in the W. pigra genome, more than those from H. manillensis (N = 3).…”

“…The destabilases and GGTs, but not the LCI, had a signal peptide. Moreover, the catalytic residues, histidine of destabilases [52], and threonine of GGTs [53] from both the W. pigra and H. manillensis were conserved.…”

“…The destabilases and GGTs, but not the LCI, had a signal peptide. Moreover, the catalytic residues, histidine of destabilases [52], and threonine of GGTs [53] from both the W. pigra and H. manillensis were conserved. In order to show the overall expression level of each antithrombotic gene family for each sample, we summed the TPM of all members in each family (tTPM).…”