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Biology subjects

Ilari, L.

Publications and source records attributed to Ilari, L..

2 recordsLinked to original sources

A quantitative interactome map reveals affinity tailored hijacking of human cellular functions by resident and pathogenic bacteria

Bacteria employ molecular mimicry to hijack host cellular functions for their own benefit. Despite the wealth of data mapping human-bacteria interactions, a comprehensive view of how these networks are fine-tuned remains elusive. In particular, there have been no large-scale screenings measuring human-bacterial protein-protein binding affinities to explore their role in functional and pathological outcomes. Here, we report the largest quantitative interactome to date between the family of human PDZ domains and C-terminal PDZ-binding motifs (PBMs) in effectors from pathogenic and resident gut bacteria. We show that bacteria tailor effector sequences to bind PDZ domains with similar or higher binding affinities than human PBMs to hijack their associated cellular functions. For a subset of the dataset, we validate PDZ-PBM interactions with the corresponding full-length proteins and show that for pathogenic E. coli effector NleA, its C-terminal PBM is necessary for interactions with proteins involved in intracellular transport and membrane organization, probably impacting the host glycocalyx to favor infection. Finally, we show that motifs overlapping with effector PBMs target interaction modules other than PDZ domains, adding an additional layer of complexity to the host-microbe molecular dialogue.

systems biology↗

Engineering SIRPα conformational plasticity to reveal a cryptic pocket suitable for structure-based drug design

The protein-protein interaction between Signal Regulatory Protein alpha (SIRP) and CD47 is a critical immune checkpoint that enables tumor immune escape, making it a key target for cancer immunotherapy. While antibody-based therapies exist, the development of small-molecule inhibitors has been hindered by the flat, featureless binding interface. Here, we report the discovery of a novel, druggable cryptic pocket within the SIRP D1 domain (the WYF pocket), revealed through a structure-based fragment screening campaign using x-ray crystallography. This pocket, defined by residues Trp38, Tyr50, and Phe74, is only accessible in a conformation that is incompatible with CD47 binding, making it a candidate for structure-based drug design and immune checkpoint inhibitor development. Through a combination of NMR spectroscopy, molecular dynamics simulations, and biophysical assays, we demonstrate that access to this cryptic site is dynamically controlled by a single "gatekeeper" residue, Gln52. The rotameric state of Gln52 dictates a conformational equilibrium between a "closed," state and a ligand-accessible "open" state. We validated this mechanism by engineering SIRP mutants to bias this equilibrium. A Q52F mutation locked the pocket in a closed state, abolishing both CD47 and fragment binding, while Q52A and Q52R mutations biased the protein toward an open state. These "open-biased" mutants not only exhibited decreased affinity for CD47 but also significantly improved binding to small-molecule fragments that inhibit the SIRP-CD47 interaction. This work reveals the intrinsic conformational plasticity of SIRP and establishes a validated structure-based roadmap for a new class of allosteric inhibitors. This flexibility-for-inhibition strategy functions by trapping a non-binding conformation and represents a broadly applicable framework for targeting this and other challenging immune checkpoints.

biochemistry↗