Molecular interactions between the VirD4 coupling protein and T4SS effectors

• Grant number: 25/16011-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2025
• End date: August 31, 2026
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Roberto Kopke Salinas
• Grantee: Davi Marcos Cintra Barbosa
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Bacteria use a variety of secretion systems to transport macromolecules across the bacterial cell envelope. Type IV Secretion Systems (T4SS) translocate proteins or protein-DNA complexes into a target cell. T4SSs may be conjugative, specialized in the transfer of plasmid DNA and, hence, involved in the spread of antibiotic resistance, or effector translocators, which are found in a variety of pathogenic bacteria. The Xanthomonas citri T4SS (T4SS-XAC) is specialized in contact-dependent injection of toxins into competing bacteria of different species. Therefore, T4SS-XAC is part of the bacteria warfare arsenal. It consists of 11 subunits (VirB1 - VirB11) and VirD4, a membrane-associated hexameric ATPase that recruits substrates for secretion. Little is known about the mechanisms behind T4SS substrate recruitment and translocation, but all T4SS-XAC substrates (so-called X-Tfes) contain a small (~ 120 aa) and highly flexible C-terminal domain called Xanthomonas VirD4 Interacting Protein Conserved Domain (XVIPCD), which binds tightly to the VirD4 All-Alpha-Domain (VirD4-AAD). We solved the NMR structure of the XAC2609 XVIPCD (XVIPCD-XAC2609) and obtained crystals of the complex between the XVIPCD-XAC2609 and VirD4-AAD, which diffracted up to 2.8 Å. However, to understand the substrate translocation mechanism it will be important to use a better model corresponding to the full-length VirD4 in the hexameric state. As an attempt to obtain samples of the hexameric VirD4, we will design several constructs by deleting the N-terminal segment that contains the transmembrane helix. We aim to obtain samples of hexameric VirD4 that are soluble and suitable for NMR titration experiments with 15N-labeled XVIPCD samples. We expect that this work will advance our understanding of T4SS-XAC substrate recruitment and translocation mechanisms in atomic detail. This knowledge will ultimately help the development of new strategies to fight bacterial antibiotic resistance and infections caused by pathogenic bacteria. (AU)