Biology subjects

Wong, K.-B.

Publications and source records attributed to Wong, K.-B..

5 recordsLinked to original sources

bioRxiv2025

Structural insights into how metallochaperones UreE and UreG interact to deliver a toxic metal to urease

Maturation of urease, a virulence factor for Helicobacter pylori infection, requires the delivery of nickel ions to the metalloenzyme. To avoid cytotoxicity, nickel ions are transferred within protein complexes of metallochaperones to ensure that the toxic metal do not escape into the cytoplasm. In the urease maturation pathway, metallochaperone UreG receives its nickel ions by forming a 2:2 complex with another metallochaperone UreE. Using C-terminal truncation variants of UreE [UreE(1-158) and UreE(1-148)], we determined the crystal structures of the UreE2G2 complex bound with nickel ions and GMPPNP, a non-hydrolysable GTP analogue. UreE-UreG interactions are asymmetric, where the UreG dimer mainly interacts with the proximal UreE. GTP binding induces conformational changes in the G2 and CPH motifs of distal UreG that enable its anchorage to the UreE2G2 complex. His68 of distal UreG moves towards and chelates a nickel ion at the UreE binding site. Formation of the UreE2G2 complex juxtaposes the nickel binding sites of UreE and UreG. Nickel transfer from UreE to UreG induces conformational changes that weaken UreE-UreG interactions, thereby facilitating UreG dissociation. We further demonstrated that the hydrogenase maturation factor HypA, providing the nickel source, could activate urease in vitro through protein-protein interaction with wild-type UreE but not with its truncation variants. UreG binding induces conformational changes in the C-terminal tail of UreE, promoting dissociation of HypA-UreE complex. Our work presents a paradigm on how GTP and nicking binding allosterically regulate the formation of a metallochaperone complex to facilitate nickel transfer. SignificanceOur work provides insights into how cells solve the problem of trafficking a toxic metal, nickel, to the active site of urease. Colonization of Helicobacter pylori in acidic human stomach requires the biosynthesis of active urease, which involves the delivery of the toxic nickel ions to the active site of the metalloenzyme. To avoid cytotoxicity, nickel ions are transported from one metallochaperone to another via the formation of protein complexes so that the toxic metal ions do not escape into the cytoplasm. Supported by structural and biochemical evidence, we present a paradigm on how GTP and nickel binding allosterically promote the formation of a complex between metallochaperones UreE and UreG to facilitate nickel transfer between the two proteins.

biochemistry↗

bioRxiv2024

The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking

Receptor-homology-transmembrane-RING-H2 (RMR) sorting receptors are essential for directing soluble cargo proteins to protein storage vacuoles in plants. These type I integral membrane proteins comprise a single transmembrane domain, an N-terminal lumenal region containing a protease-associated domain for cargo recognition, and a C-terminal cytoplasmic region (CT) with a Really-Interesting-New-Gene-H2 (RING-H2) domain. Here, we determined the crystal structure of the RING-H2 domain of Arabidopsis RMR isoform-1 (AtRMR1-RING), where the conserved C3H2C3 motif coordinates two Zn ions, a feature typical of RING-type E3 ligases. AtRMR1-RING was shown to interact with Arabidopsis E2 ubiquitin-conjugating enzyme, and exhibits E3 ligase activity in an in vitro ubiquitination assay. Biochemical analysis reveals that I234Y substitution disrupted the E2/E3 interaction and greatly reduced E3 ligase activity. Furthermore, we showed that the conserved RING-H2 domains of AtRMR isoform 2, 3 and 4 are also E3 ligases. Inactivation of E3 ligase activity by the I234Y mutation resulted in Golgi retention of AtRMR1-CT and AtRMR2. These findings suggest that the E3 ligase activity is essential for post-Golgi trafficking of RMR receptors, providing new insights into receptor-mediated protein sorting in plants.

plant biology↗

bioRxiv2024

Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance

Macroautophagy (hereafter autophagy) is essential for cells to respond to nutrient stress by delivering cytosolic contents to vacuoles for degradation via the formation of a multi-layer vesicle named autophagosome. A set of autophagy-related (ATG) regulators are recruited to the phagophore assembly site for the initiation of phagophore, as well as its expansion and closure and subsequent delivery into the vacuole. However, it remains elusive that how the phagophore assembly is regulated under different stress conditions. Here, we described an unknown Arabidopsis (Arabidopsis thaliana) cytosolic ATG8-interaction protein family (ERC1/2), that binds ATG8 and NBR1 to promote autophagy. ERC1 proteins translocate to the phagophore membrane and develop into classical ring-like autophagosomes upon autophagic induction. However, ERC1 proteins form large droplets together with ATG8e proteins when in the absence of ATG8 lipidation activity. We described the property of these structures as phase-separated membraneless condensates by solving the in vivo organization with spatial and temporal resolution. Moreover, ERC1 condensates elicits a strong recruitment of the autophagic receptor NBR1. Loss of ERC1 suppressed NBR1 turnover and attenuated plant tolerance to heat stress condition. This work provides novel insights into the mechanical principle of phagophore initiation via an unreported ERC1-mediated biomolecular condensation for heat tolerance in Arabidopsis.

plant biology↗

bioRxiv2023

A converged ubiquitin-proteasome pathway for the degradation of TOC and TOM tail-anchored receptors

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon at the outer membranes of chloroplasts) and the TOM (translocon at the outer membranes of mitochondria) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA+ ATPase CDC48, after which a previously characterized cytosolic protein, TTOP, binds to the exposed transmembrane domains (TMDs) at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

plant biology↗

bioRxiv2020

Simeprevir suppresses SARS-CoV-2 replication and synergizes with remdesivir

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

microbiology↗