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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: TOXINS; v. 17, n. 3, p. 17-pg., 2025-02-27.

Resumo

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)

• Processo FAPESP: 24/01956-5 - Integração da Flexibilidade Proteica em Modelos de Aprendizado de Máquina para Identificação Virtual de Hits
• Beneficiário: Raghuvir Krishnaswamy Arni
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 20/08615-8 - Exosítios de proteínas, sítios crípticos e moonlighting: identificação, mapeamento funcional e efeitos de alteração estrutural
• Beneficiário: Raghuvir Krishnaswamy Arni
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 24/13327-2 - Integração da Flexibilidade Proteica em Modelos de Aprendizado de Máquina para Identificação Virtual de Hits
• Beneficiário: Jorge Enrique Hernández González
• Modalidade de apoio: Bolsas no Brasil - Programa Fixação de Jovens Doutores
• Processo FAPESP: 24/16184-8 - Exosítios de proteínas, sítios crípticos e moonlighting: identificação, mapeamento funcional e efeitos de alteração estrutural
• Beneficiário: Daniel Zocchi Doherty
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 24/00876-8 - Exosítios de proteínas, sítios crípticos e moonlighting: identificação, mapeamento funcional e efeitos de alteração estrutural
• Beneficiário: Carolina Gismene
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado