Biology subjects

Trapani, S.

Publications and source records attributed to Trapani, S..

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↗

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

Structure of the vasopressin hormone-V2 receptor-β-arrestin1 ternary complex

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with {beta}-arrestin1. It reveals an atypical position of {beta}-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/{beta}-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R C-terminus are clearly identified and interact extensively with the {beta}-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a striking structural variability among GPCR-arrestin signaling complexes.

biochemistry↗

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↗

bioRxiv2019

In situ structure of rotavirus VP1 RNA-dependent RNA polymerase

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the \"double-layer particle\" or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions (\"triple-layer particles\" or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

molecular biology↗

bioRxiv2019

MglA functions as a three-state GTPase to control movement reversals of Myxococcus xanthus

In Myxococcus xanthus, directed movement is controlled by inter-dependent pole-to-pole oscillations of the small GTPase MglA, its GAP MglB and the RomR protein. However, these proteins have strikingly different oscillatory regimes such that MglA is segregated from MglB and RomR at reversal activation. The molecular mechanism whereby information is exchanged between the lagging and leading poles resulting in MglA detachment from the leading pole during reversals has remained unknown. Here, we show that MglA has two GTP-bound forms, one of which is insensitive to MglB (MglA-GTP*) and is re-sensitized to MglB by a feedback mechanism operated by MglA-GDP. By identifying the region of MglB that is critical for its association to the lagging pole, we demonstrate that MglA-GTP* is functional in vivo. These data suggest that MglA-GDP acts as a soluble messenger to convert polar MglA-GTP* into a diffusible MglA-GTP species, explaining MglA re-localization to the opposite pole during reversals.

microbiology↗