The diguanylate cyclase YfiN is a central regulator in the adaptive persistence of Pseudomonas aeruginosa within the cystic fibrosis lung, where it drives the formation of small colony variants (SCVs) through sustained production of cyclic di-GMP (c-di-GMP). As part of the tripartite YifBNR signaling module, YfiN converts periplasmic stimuli into cytosolic responses via an allosteric inside-out mechanism. Despite its clinical significance, the molecular basis of this regulation remained unknown due to the lack of structural data. Here, we present the first high-resolution crystal structure of the catalytic GGDEF domain of YfiN at 2.8 Å resolution, revealing key insights into its activation mechanism.
The YfiNGGDEF domain adopts a canonical fold consisting of a five-stranded β-sheet core (strands 2–3–1–6–7) surrounded by five α-helices (A–F), closely resembling other known GGDEF domains such as PleD and WspR. A prominent peripheral β-hairpin (strands 4–5) is conserved across homologues, except in WspR, which displays a divergent loop conformation. However, a defining feature lies in the allosteric inhibitory site (I-site), which is structurally degenerated. While the primary I-site (Ip) retains the conserved RxxD motif essential for catalysis, the secondary I-site (Is) lacks critical residues required for dimeric c-di-GMP binding. Specifically, Arg-273 is replaced by Asp, and the functionally important arginine analogous to Arg-390 in PleD is substituted with Asn-351. Moreover, the α-A helix containing Is is displaced due to steric hindrance from Tyr-379, effectively collapsing the I-site cavity.
This structural degeneration abolishes the ability of YfiNHAMP-GGDEF to bind c-di-GMP, as confirmed by isothermal titration calorimetry (ITC), which detected no heat change upon titration with the product. In contrast, the enzyme binds GTP with high affinity (Kd ≈ 0.18 µM), indicating that substrate recognition remains intact. Real-time enzymatic assays using circular dichroism spectroscopy show complete conversion of GTP to c-di-GMP, confirming robust catalytic activity.
Notably, a truncated construct lacking the HAMP domain (YfiNGGDEF) binds GTP with similar affinity yet displays no detectable activity, demonstrating that the HAMP domain is indispensable for catalysis.4-tert-Amylphenol MedChemExpress Analytical ultracentrifugation confirms that YfiNHAMP-GGDEF exists as a monomer in solution (S20,w = 2.3), yet remains catalytically active. This paradox suggests that transient dimerization occurs during catalysis, likely driven by conformational changes induced by substrate binding or allosteric signals. The observed sigmoidal kinetic profile further supports cooperative behavior, implying that the transition from inactive monomer to active dimer is regulated and not spontaneous.
Homology modeling of full-length YfiN, based on fold prediction and structural templates including LapD from P. fluorescens, proposes a V-shaped, domain-swapped architecture for the N-terminal periplasmic region. In this model, YfiR binding at the central gorge induces a bending motion that propagates through the transmembrane helices and HAMP domain. This torsional shift is transmitted via the linker region (residues 247–253) to the GGDEF domains, unlocking them from an inactive state and enabling dimer formation. The linker region acts as a molecular hinge whose conformation is controlled by the HAMP domain, thereby regulating access to the active site.Rabbit Anti-Human IgG H&L custom synthesis
Sequence conservation analysis across orthologs highlights highly preserved regions in the periplasmic domain, HAMP bundle, and linker loop—key elements in signal transmission and catalytic activation.PMID:34920288 Mutations identified in clinical isolates map precisely to these functional sites, validating their role in vivo. The deletion mutant Δ255–257 is inactive and dominant over activating substitutions, underscoring the functional importance of this linker region.
These findings collectively support a mechanistic model in which YfiN functions as an ON/OFF switch, activated exclusively by external signals sensed through YfiR. The absence of product feedback inhibition ensures that once activated, c-di-GMP production proceeds without self-limitation, promoting long-term persistence in hostile host environments. This design allows P. aeruginosa to maintain biofilm formation and antibiotic resistance under chronic infection conditions.
This study provides a structural and functional framework for understanding how periplasmic signals are transduced into cytosolic responses in bacterial signaling systems. Targeting the HAMP-linker interface or stabilizing inactive conformations could offer novel therapeutic strategies to disrupt biofilm formation in chronic P. aeruginosa infections. Given the rising threat of antimicrobial resistance, elucidating such allosteric mechanisms opens new avenues for precision interventions against persistent pathogens.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com