Structural and Energetic Evidence Supports the Non-Covalent Phosphate Cyclization by the Class II Phospholipase D from Loxosceles intermedia

Author(s)

Gismene, Carolina ; Bachega, Jose Fernando Ruggiero ; Doherty, Daniel Z. ; Veiga, Silvio Sanches ; Arni, Raghuvir K. ; Gonzalez, Jorge Enrique Hernandez

• Document type: Journal article
• Source: TOXINS; v. 17, n. 3, p. 17-pg., 2025-02-27.

Abstract

Phospholipase D (PLD) enzymes from Loxosceles spider venom mediate envenomation pathology by cleaving phospholipid headgroups. We revisited the crystal structure of Loxosceles intermedia PLD (PDB: 3RLH) to evaluate two alternative mechanisms-covalent and non-covalent-for headgroup cleavage. The covalent mechanism involves a nucleophilic attack on the substrate's P atom by catalytic histidine, forming a phosphohistidine intermediate. It was originally suggested that this intermediate hydrolyzes, leading to linear phosphates. The non-covalent mechanism relies on the substrate's hydroxyl group performing an intramolecular attack on the P atom, thereby generating a cyclic phosphate. Structural refinement of the crystal structure revealed a cyclic phosphate bound at the active site, replacing previously assigned PEG molecules. This cyclic product, stabilized by His12, His47, and Mg2+, provides structural evidence that supports phosphate cyclization. The results of computational analyses, including molecular dynamics and quantum mechanics/molecular mechanics simulations, further support the non-covalent mechanism as the energetically preferred pathway, with a significantly lower activation barrier. Our findings highlight the role of substrate orientation and of the catalytic His residues in transphosphatidylation, advancing our understanding of PLD enzymology and providing insights for the design of inhibitors against Loxosceles envenomation. (AU)

• FAPESP's process: 24/01956-5 - Integrating Protein Flexibility into Machine Learning Models for Virtual Hit Identification
• Grantee: Raghuvir Krishnaswamy Arni
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/08615-8 - Protein exosites, cryptic sites and moonlighting: identification, functional mapping and effects of changes in structure
• Grantee: Raghuvir Krishnaswamy Arni
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 24/13327-2 - Integrating Protein Flexibility into Machine Learning Models for Virtual Hit Identification
• Grantee: Jorge Enrique Hernández González
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• FAPESP's process: 24/16184-8 - Protein exosites, cryptic sites and moonlighting: identification, functional mapping and effects of structural alteration.
• Grantee: Daniel Zocchi Doherty
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 24/00876-8 - Protein exosites, cryptic sites and moonlighting: identification, functional mapping and effects of changes in structure
• Grantee: Carolina Gismene
• Support Opportunities: Scholarships in Brazil - Post-Doctoral