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ARS Home » Plains Area » Fort Collins, Colorado » Center for Agricultural Resources Research » Soil Management and Sugarbeet Research » Research » Publications at this Location » Publication #436864
Research Project: Genomic Mining of Sugar Beet Crop Wild Relative Germplasm Resources for New Sources of Disease Resistance

Location: Soil Management and Sugarbeet Research

Title: Structural characterization the LlaI anti-phage defense system reveals insights into the evolution of nucleotide specificity and the organization of DNA binding in McrBC restriction complexes

Author
item BUI, ANTHONY - Cornell University
item HOSFORD, CHRISTOPHER - Cornell University
item NIU, YIMING - Cornell University
item SANTIAGO, EMERSON - Cornell University
item WAGNER, MATEUSZ - Cornell University
item Chappie, Joshua

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 9/1/2026
Publication Date: 9/1/2026
Citation: Bui, A.Q., Hosford, C.J., Niu, Y., Santiago, E., Wagner, M.M., Chappie, J.S. 2026. Structural characterization the LlaI anti-phage defense system reveals insights into the evolution of nucleotide specificity and the organization of DNA binding in McrBC restriction complexes. bioRxiv. https://doi.org/10.64898/2026.08.31.748284.
DOI: https://doi.org/10.64898/2026.08.31.748284

Interpretive Summary: Antibiotic resistance is a significant global health threat affecting disease management in humans, livestock, and agriculture. Bacteriophage viruses that selectively infect and kill bacteria represent a promising clinical alternative. Despite recent success treating multidrug resistant infections with bacteriophages in humans, pigs, and cattle, widespread use of phage therapy has been limited by bacterial-encoded anti-phage defense systems, many of which remain poorly characterized. Using X-ray crystallography, protein biochemistry, and structural modeling, scientists from USDA and Cornell elucidated the architecture and biochemical properties an anti-phage defense system from Lactococcus lactis, specifically showing how this protein complex binds and cleaves DNA. The structural findings illustrate how small molecules could be used to inhibit the activity of this defense machinery, which provides a generalizable strategy to increase the efficacy of bacteriophages and broaden their use. This work also has important implications for the design of novel, programmable nucleases that can serve as valuable diagnostic tools.

Technical Abstract: Canonical McrBC enzymes are nucleotide-powered, motor-driven endonucleases that bind and cleave modified bacteriophage DNA. Non-canonical McrBC homologs like LlaI and BsuMI are distinguished by a unique three-gene organization and the ability to target DNA site-specifically. Here, we report the atomic-resolution crystal structures of the DNA-binding module LlaI.R1 and AAA+ motor LlaI.R2 from the Lactococcus lactis LlaI anti-phage defense system. The crystallized LlaI.R2 hexamer traps two distinct active site conformations that correlate to different states of the nucleotide hydrolysis cycle and reveal that the organization of the critical catalytic machinery present in canonical McrB homologs is also conserved in non-canonical R2 proteins. Although canonical McrB homologs are strictly GTP-specific, we find that the R2 proteins from LlaI and BsuMI do not discriminate between different nucleotides, even when in complex with their respective R1 partners. Crystallographic analysis of LlaI.R1 identifies a potential DNA-binding surface as well as a putative interface for interaction with LlaI.R2. Computational docking and mutational analysis support our modeling of the LlaI.R1:R2 complex. Together, our data provide new insights into the evolution of nucleotide specificity and the organization of DNA binding in McrBC restriction complexes.