Science » Publications and News

The structure of the mammalian bornavirus polymerase complex

Mon, 29 Sep 2025 16:03:00 GMT

Borna disease virus 1 (BoDV-1) is a non-segmented RNA virus with one of the smallest known RNA virus genomes. BoDV-1 replicates in the nucleus of infected cells using a virally encoded polymerase complex composed of the large protein and phosphoprotein. Here, we present the BoDV-1 polymerase complex at resolutions up to 2.8 Å, describing the fully ordered large polymerase protein bound to tetrameric phosphoprotein. The complex is maintained through the ordered C-terminal region of one copy of the phosphoprotein. Analysis of the model reveals a conserved methyltransferase domain, though key S-adenosyl methionine binding residues are missing. While no RNA is observed in our models, analysis of a sample under reaction conditions induces an opening and closing of the template entry and exit channels, respectively. Higher-order polymerase assemblies suggest oligomerisation as a conserved feature of negative strand RNA virus polymerases. We provide a molecular framework to investigate bornavirus replication and transcription.

Molecular Basis for Short-Chain Thioester Hydrolysis by Acyl Hydrolases in trans-Acyltransferase Polyketide Synthases

Tue, 27 May 2025 09:26:00 GMT

Polyketide synthases (PKSs) are multidomain enzymatic assembly lines that biosynthesize a wide selection of bioactive natural products from simple building blocks. In contrast to their cis-acyltransferase (AT) counterparts, trans-AT PKSs rely on stand-alone ATs to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. Trans-AT PKS gene clusters also encode stand-alone acyl hydrolases (AHs), which are predicted to share the overall fold of ATs but function like type II thioesterases (TE_IIs), hydrolyzing aberrant acyl chains from ACP domains to promote biosynthetic efficiency. How AHs specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AHs and ACP domains contributes to recognition of cognate and noncognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the construction of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by trans-AT PKSs, and provide foundations for future bioengineering efforts to offload biosynthetic intermediates or enhance product yields.

Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport

Fri, 06 Dec 2024 16:35:24 GMT

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2

Thu, 28 Nov 2024 15:40:00 GMT

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO₂. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO₂. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.