Biology subjects

Lai-Kee-Him, J.

Publications and source records attributed to Lai-Kee-Him, J..

6 recordsLinked to original sources

bioRxiv2024

Structure of the turnip yellows virus particles. How is the readthrough domain exposed on the capsid surface?

Turnip yellows virus (TuYV) is a plant virus infecting important crops such as oilseed rape. TuYV is phloem-restricted and transmitted by aphids. The capsid contains two subunit types: the major capsid protein (CP) and a minor component (RTP*) which arises from the C-terminal cleavage of a readthrough product (RTP). RTP* contains the CP sequence fused with a structured domain, denoted NRTD, which is a key determinant of virus transmission. Though both CP and RTP* are involved in virus movement and aphid transmission, how RTP* is incorporated into the capsid is poorly understood. We present here the structural characterisation, by immunogold labelling and 3D cryo-EM, of the wild-type TuYV and a mutant whose capsid contains the CP only. We show that incorporation of RTP* does not impair the capsid structure, and the NRTD does not adopt well-defined positions at the capsid surface. The number of incorporated RTP*s suggests a random insertion.

microbiology↗

bioRxiv2023

In situ characterization of late Chikungunya virus replication organelle

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculo-skeletal disease in humans. The viral RNA genome replication occurs in membrane spherules named replication organelles (ROs). In this work, we investigate the native structural organization of CHIKV ROs in their cellular context using in situ cryogenic-electron microscopy approaches at late replication stage. We observed previously unreported diameter heterogeneity of ROs at the plasma membrane of infected human cells. CHIKV ROs were only marginally detected in cytopathic vacuoles where they are homogeneous in size, suggesting a finely regulated internalization process. Our data show that ROs maintained at the plasma membrane beyond the first viral cycle are dynamically active both in viral RNA replication, in its export to the cell cytosol, but also in the production of viral proteins. We suggest that late CHIKV ROs have an amplifying role or represent an alternative pathway in the production of infectious viral particles. All these observations bring new insight into the CHIKV life cycle in human cells.

molecular biology↗

bioRxiv2022

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response {sigma}B factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that {sigma}B is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results revealed that nonconserved region of {sigma} is an allosteric controller of transcription initiation and demonstrated how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

molecular biology↗

bioRxiv2022

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR complex

SummaryLiving organisms have developed protein sensors helping them to adapt to their environment1. The aryl hydrocarbon receptor (AHR) is an emblematic member of this class of proteins, and a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites2. However, in the absence of high-resolution structural data, a molecular understanding of how AHR is activated by such diverse compounds is lacking. Here we present a 2.85 [A] cryo-electron microscopy structure of the cytosolic complex comprising AHR bound to the ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2. The structure reveals a closed Hsp90 dimer with AHR threaded through its lumen. XAP2 directly interacts with Hsp90 and the AHR ligand-binding domain, thereby acting as a brace stabilizing the entire complex. Importantly, we provide the first experimental visualization of the AHR PAS-B domain bound to a ligand, revealing a unique organization of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing unprecedented structural details of the molecular initiating event leading to AHR activation, our study rationalizes prior biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

biochemistry↗

bioRxiv2021

Cryo-EM structure of the transcription termination factor Rho from Mycobacterium tuberculosis reveals mechanism of resistance to bicyclomycin

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. Here, we address these unusual features by solving the cryo-EM structure of MtbRho at 3.3 [A] resolution, providing a new framework for future antibiotic development. The MtbRho hexamer is poised into a pre-catalytic, open-ringed state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP {gamma}-phosphates, and confers resistance to BCM at the expense of motor efficiency.

molecular biology↗

bioRxiv2020

Structure of the antidiuretic hormone vasopressin receptor signalling complex.

Arginine-vasopressin (AVP) is a neurohypophysial peptide known as the antidiuretic hormone. It forms an active signalling complex with the V2 receptor (V2R) and the Gs protein, promoting a cAMP/PKA-dependent aquaporin insertion in apical membranes of principal cells of the renal collecting ducts and ultimately, water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signalling system are still unknown. To fill this gap of knowledge, we report here the structure of the AVP-V2R-Gs complex using cryo-electron microscopy (cryo-EM). Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and NMR spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes and could thus represent distinct complex conformations along the signalling activation pathway. Importantly, as compared to those of other class A GPCR-Gs complexes, the structures revealed an original receptor-Gs interface in which the Gs subunit penetrates deeper into the active V2R, notably forming an ionic bond between its free C-terminal carboxylic function and the side chain of R137 in the V2R. Interestingly, the structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases with opposite clinical outcomes, cNDI or NSIAD respectively. Our study thus provides important structural insights into the function of this clinically relevant GPCR signalling complex.

biochemistry↗