Biology subjects

Huang, R. H.

Publications and source records attributed to Huang, R. H..

6 recordsLinked to original sources

bioRxiv2026

Cyclase-generated 3,5-cGMP activates PucsarTIR antiphage defense through a stepwise zig-zag aligned filament assembly

Bacteria use nucleotide second messengers to couple phage recognition to antiviral effector activation. Here, we identify Pucsar, an ofshoot of the Pycsar-related antiphage family, whose cyclases generate 3,5-cGMP from GTP, that are associated with Toll/Interleukin-1 receptor (TIR) and transmembrane (TM) effectors. In the PucsarTIR system, cGMP produced by cyclase PucC, selectively activates the cognate PucTIR NADase. X-ray crystallography shows that PucC adopts a dimeric cyclase scaffold with a guanine-compatible base-recognition pocket. Cryo-EM and mutational analyses revealed that apo-PucTIR, composed of cyclic nucleotide binding and TIR domains, is self-sequestered as an inactive tetramer, in which TIR domains are proximal but misoriented. Sequential cGMP binding remodels this assembly through partially occupied intermediates and stabilizes a fully cGMP-bound dimer, that polymerizes into a zig-zag aligned filament, creating composite NAD+-binding pockets required for efficient NADase activity. These findings establish 3',5'-cGMP as an antiphage second messenger and reveal how cyclic purine signaling converts self-sequestered TIR effectors into a NADase-competent filament.

microbiology↗

bioRxiv2025

Biochemical and structural characterization of the RlaP family nucleotidyltransferase potentially involved in RNA repair

Nucleotidyltransferases (NTases) of Pol{beta} superfamily are highly diverse and play many important biological functions. However, many families of NTases remain uncharacterized. Because of physical proximity of the genes encoding NTases of PF10127 family to those encoding RNA ligases involved in RNA repair, PF10127 family of NTases was named RlaP (RNA ligase-associating Pol{beta}). Here we report comprehensive characterization of two RlaP from Pseudomonas fluorescens and Pseudomonas aeruginosa (PfRlaP and PaRlaP), respectively. Our study showed that, among macromolecules isolated from E. coli cells, only RNAs are the substrates of RlaP. In vitro assays employing synthetic RNAs as substrates demonstrated that RlaP catalyzes addition of one or two nucleotide monophosphate (NMP) to the 3-hydroxyl group of RNAs, with the damaged RNAs as the preferred substrates. The crystal structure of PfRlaP provided insight into molecular recognition of RNA substrate and nucleotide triphosphate (NTP) by RlaP. Aminoacylation assays indicate that RlaP is required to restore biological function of the repaired tRNAs that have suffered excessive damage. Based on our studies, we propose an in vivo scenario where cell survival requires the involvement of RlaP in RNA repair to restore the biological function of the damaged RNAs.

biochemistry↗

bioRxiv2025

A Pipeline for Screening Small Molecule-Enhanced Protein Stability in A Bacterial Orphan Receptor

Bacterial one-component signaling proteins integrate sensory and gene regulation functions within the same polypeptide, creating powerful natural sensors of environmental conditions which can also be adapted into powerful tools for synthetic biology and biotechnology. A key sensor motif within many of these proteins is the Per- ARNT-Sim (PAS) domain, known for its conserved fold yet highly divergent sequences, allowing for a broad range of ligands to control PAS protein function by changes in small molecule binding occupancy or configuration. This diversity introduces a challenging step - identification of ligands for "orphan" PAS proteins which show signatures of ligand binding but no copurifying high-affinity bound small molecules - into characterization and engineering of such proteins. In this study, we characterized CU228, a putative PAS-HTH transcription factor from Candidatus Solibacter usitatus, as a novel model system for searching for novel ligands by small molecule stabilization. Bioinformatics and structural analyses predicted a PAS domain with an Trp-rich internal cavity, suggesting potential small molecule interactions. Using a [~]760 compound fragment library, differential scanning fluorimetry identified three ligands (KG-96, KG- 408, and KG-484) that substantially increased CU228 thermal stability with {Delta}Tm values up to +10{degrees}C. Microfluidic modulation spectroscopy (MMS) revealed ligand-induced preservation of -helical and {beta}-sheet integrity under thermal stress. Saturation transfer difference NMR confirmed direct binding of all three ligands and enabled estimation of micromolar-range dissociation constants, consistent with expected fragment-level affinity. Our findings expand the analytical toolbox for probing protein-ligand interactions in flexible, signal-responsive systems, laying the groundwork for designing synthetic chemogenetic variants of one-component transcription factors.

biochemistry↗

bioRxiv2025

Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation

In bacteria, spontaneous mRNAs degradation and ribotoxin-induced RNA damage are two main biological events that lead to the stall of protein translation. The ubiquitous trans-translation system as well as several alternative rescue factors (Arfs) are responsible for rescuing the stalled ribosomes caused by truncated mRNAs that lack the stop codons. To date, protein release factor homolog (PrfH) is the only factor known to rescue the stalled ribosome damaged by ribotoxins. Here we show that a subfamily of PrfH, exemplified by PrfH from Capnocytophaga gingivalis (CgPrfH), rescues both types of stalled ribosomes described above. Our in vitro biochemical assays demonstrate that CgPrfH hydrolyzes the peptides attached to P-site tRNAs when in complex with both the damaged and intact ribosomes. Two cryo-EM structures of CgPrfH in complex with the damaged and intact 70S ribosomes revealed that CgPrfH employs two different regions of the protein to recognize two different stalled ribosomes to orient the GGQ motif for peptide hydrolysis. Thus, using a combination of bioinformatic, biochemical, and structural characterization described here, we have uncovered a family of ribosome rescue factors that possesses dual activities to resolve two distinct stalled protein translation in bacteria.

biochemistry↗

bioRxiv2021

Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB

In bacteria, rescue of stalled ribosomes due to 3-truncated mRNAs is carried out by the ubiquitous trans-translation system as well as alternative ribosome-rescue factors such as ArfA and ArfB. It is unclear, however, how the stalled ribosomes caused by ribosomal damages are rescued. Here, we report that a bacterial system composed of PrfH and RtcB not only rescues a stalled ribosome resulting from a specific damage in the decoding center but also repairs the damage afterwards. Peptide release assays reveal that PrfH is only active with the damaged ribosome, but not with the intact one. A 2.55-angstrom cryo-EM structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into specific recognition of the damage site by PrfH. RNA repair assays demonstrate that PrfH-coupled RtcB efficiently repairs the damaged 30S ribosomal subunit, but not the damaged tRNAs. Thus, our studies have uncovered a biological operation by a pair of bacterial enzymes, aiming to reverse the potentially lethal damage inflicted by an invading ribotoxin for cell survival.

biochemistry↗

bioRxiv2021

Molecular mechanisms of the CdnG-Cap5 antiphage defense system employing 3',2'-cGAMP as the second messenger

Cyclic-oligonucleotide-based antiphage signaling systems (CBASS) are diverse and abundant in bacteria. Here, we present biochemical and structural characterization of two CBASS systems, composed of CdnG and Cap5, from Asticcacaulis sp. and Lactococcus lactis. We show that CdnG from Asticcacaulis sp. synthesizes 3,2-cGAMP in vitro, and 3,2-cGAMP is the biological signaling molecule that activates Cap5 for DNA degradation. Crystal structures of Cap5, together with the SAVED domain in complex with 3,2-cGAMP, provide insight into the architecture of Cap5 as well as molecular recognition of 3,2-cGAMP by the SAVED domain of Cap5. Amino acid conservation of the SAVED domain of Cap5, together with mutational studies, led us to propose a novel mechanism of Back-to-Front stacking of two SAVED domains, mediated by 3,2-cGAMP, to activate HNH nuclease domain for DNA degradation. Our study of the most abundant CBASS system provides new insight into mechanisms employed by bacteria in their conflicts against phage.

biochemistry↗