Park, J. B. | Search bioRxiv

bioRxiv2026

Host membrane cholesterol constrains constitutive signaling of the oncogenic KSHV GPCR ORF74

Cholesterol is a major structural component of the plasma membrane and a key allosteric regulator of G protein-coupled receptors (GPCRs), yet its role in controlling constitutive receptor activity remains poorly understood. Virally encoded GPCRs provide an ideal system to address this question because many exhibit constitutive signaling that promotes viral persistence and pathogenesis, although how excessive receptor activation is restrained remains unknown. Here, we identify a previously unrecognized cholesterol-dependent allosteric mechanism by which the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) GPCR ORF74 constrains its constitutive activity. CryoEM structural analysis reveals a cholesterol-binding pocket formed by transmembrane helices 3, 5, and 6 that is present only in the inactive receptor. Cholesterol binding restrains the outward movement of transmembrane helix 6, stabilizes the inactive conformation, and suppresses spontaneous receptor activation, as supported by molecular dynamics simulations. Mechanistically, replacement of the canonical DRY motif with a non-canonical VRY motif exposes the conserved R1433.50 residue, creating a membrane-facing cholesterol-binding interface that couples membrane cholesterol to receptor conformational control. Consistent with this model, cellular cholesterol depletion enhances ORF74 signaling, whereas disruption of cholesterol binding impairs receptor stabilization. Together, our findings uncover a previously unrecognized mechanism by which a viral GPCR exploits host membrane cholesterol to regulate its constitutive activity, suggesting that persistent viruses optimize constitutive signaling by coupling receptor activity to host lipid-dependent allosteric regulation.

microbiology↗

bioRxiv2024

Structural basis of the catalytic and allosteric mechanism of bacterial acetyltransferase PatZ

GCN5-related N-Acetyltransferases (GNATs) play a crucial role in regulating bacterial metabolism by acetylating specific target proteins. Despite their importance in bacterial physiology, the mechanisms underlying GNATs enzymatic and regulatory functions remain poorly understood. In this study, we elucidated the structures of Escherichia coli PatZ, a type I GNAT, and investigated its ligand interactions, catalytic processes, and allosterism. PatZ functions as a homotetramer, with each subunit comprising a catalytic domain and a regulatory domain. Our findings reveal that the regulatory domain is essential for acetyltransferase activity, as it not only induces cooperative conformational changes in the catalytic domain but also directly contributes to the formation of substrate binding pockets. Furthermore, a protein structure-based analysis on the evolution of bacterial GNAT types reveals a distinct pattern of the regulatory domain across phyla, underscoring the regulatory domains critical role in responding to cellular energy status. SIGNIFICANCE STATEMENTPost-translational modifications, particularly acetylation mediated by GCN5-related N-Acetyltransferases (GNATs), play a crucial role in bacterial physiology. Protein acetyltransferase Z (PatZ) is a key GNAT with diverse substrates, essential for understanding the bacterial acetylome. This study employs cryogenic electron microscopy, X-ray crystallography, and biochemical analyses to elucidate the mechanistic regulation of Escherichia coli PatZ. Our high-resolution structures reveal PatZs homo-tetrameric architecture, with each subunit comprising regulatory and GNAT domains. We characterize ligand-PatZ interactions, demonstrating ligand-induced conformational changes that facilitate allosteric regulation of the catalytic domain. Furthermore, our analyses elucidate the regulatory domains contribution to substrate binding pocket formation, potentially enhancing substrate specificity. Structure-based phylogenetic analysis provides insights into the evolution of diverse regulatory domains in the GNAT superfamily across bacterial taxonomy. This first visualization of PatZ advances our mechanistic understanding of bacterial physiology, offering novel insights into GNAT-mediated bacterial adaptations. HIGHLIGHTS- E. coli PatZ forms a homotetramer, with each subunit possessing a GNAT catalytic domain and a regulatory domain. - Cooperative binding of acetyl-CoA to the regulatory domains is a prerequisite for inducing the structural compatibility of the catalytic domain with a substrate. - Diverse regulatory domains in GNATs evolved to adapt to varied metabolic conditions across bacterial taxonomy.

biochemistry↗