The cytoplasmic synthesis and coupled membrane translocation of eukaryotic polyphosphate by signal-activated VTC complex.

Inorganic polyphosphate (polyP) is an ancient energy metabolite and phosphate store that occurs ubiquitously in all organisms. The vacuolar transporter chaperone (VTC) complex integrates cytosolic polyP synthesis from ATP and polyP membrane translocation into the vacuolar lumen. In yeast and in other eukaryotes, polyP synthesis is regulated by inositol pyrophosphate (PP-InsP) nutrient messengers, directly sensed by the VTC complex. Here, we report the cryo-electron microscopy structure of signal-activated VTC complex at 3.0 Å resolution. Baker's yeast VTC subunits Vtc1, Vtc3, and Vtc4 assemble into a 3:1:1 complex. Fifteen trans-membrane helices form a novel membrane channel enabling the transport of newly synthesized polyP into the vacuolar lumen. PP-InsP binding orients the catalytic polymerase domain at the entrance of the trans-membrane channel, both activating the enzyme and coupling polyP synthesis and membrane translocation. Together with biochemical and cellular studies, our work provides mechanistic insights into the biogenesis of an ancient energy metabolite.

Structural insights into DNA N6-adenine methylation by the MTA1 complex.

N6-methyldeoxyadenine (6mA) has recently been reported as a prevalent DNA modification in eukaryotes. The Tetrahymena thermophila MTA1 complex consisting of four subunits, namely MTA1, MTA9, p1, and p2, is the first identified eukaryotic 6mA methyltransferase (MTase) complex. Unlike the prokaryotic 6mA MTases which have been biochemically and structurally characterized, the operation mode of the MTA1 complex remains largely elusive. Here, we report the cryogenic electron microscopy structures of the quaternary MTA1 complex in S-adenosyl methionine (SAM)-bound (2.6 Å) and S-adenosyl homocysteine (SAH)-bound (2.8 Å) states. Using an AI-empowered integrative approach based on AlphaFold prediction and chemical cross-linking mass spectrometry, we further modeled a near-complete structure of the quaternary complex. Coupled with biochemical characterization, we revealed that MTA1 serves as the catalytic core, MTA1, MTA9, and p1 likely accommodate the substrate DNA, and p2 may facilitate the stabilization of MTA1. These results together offer insights into the molecular mechanism underpinning methylation by the MTA1 complex and the potential diversification of MTases for N6-adenine methylation.

Structural analysis of the meiosis-related protein MS5 reveals non-canonical papain enhancement by cystatin-like folds.

MS5 is a meiosis-related protein belonging to the Brassicaceae-specific domain of unknown function family and characterized by the MS5 superfamily domain (MSD). In this study, we elucidated the three-dimensional crystal structure and potential biochemical function of the MSD. It was observed that the MSD adopts a cystatin-like fold, mainly consisting of a central α-helix and four- or five-stranded antiparallel ß-sheets that wrap around it. However, unlike cystatins, which inhibit cysteine proteases, the MSD displayed allosteric activation of papain. We believe that our study provides insight into novel mechanisms of proteolytic enzyme regulation and may serve as a basis for functional studies of the MS5 family proteins in plants.

Crystal structure of the bacterial acetate transporter SatP reveals that it forms a hexameric channel.

Acetate is found ubiquitously in the natural environment and can be used as an exogenous carbon source by bacteria, fungi, and mammalian cells. A representative member of the acetate uptake transporter (AceTr) family named SatP (also yaaH) has been preliminarily identified as a succinate-acetate/proton symporter in Escherichia coli However, the molecular mechanism of acetate uptake by SatP still remains elusive. Here, we report the crystal structure of SatP from E. coli at 2.8 Å resolution, determined with a molecular replacement approach using a previously developed predicted model algorithm, which revealed a hexameric UreI-like channel structure. Structural analysis identified six transmembrane (TM) helices surrounding the central channel pore in each protomer and three conserved hydrophobic residues, FLY, located in the middle of the TM region for pore constriction. According to single-channel conductance recordings, performed with purified SatP reconstituted into lipid bilayer, three conserved polar residues in the TM1 facing to the periplasmic side are closely associated with acetate translocation activity. These analyses provide critical insights into the mechanism of acetate translocation in bacteria and a first glimpse of a structure of an AceTr family transporter.

Structural basis of the arbitrium peptide-AimR communication system in the phage lysis-lysogeny decision.

A bacteriophage can replicate and release virions from a host cell in the lytic cycle or switch to a lysogenic process in which the phage integrates itself into the host genome as a prophage. In Bacillus cells, some types of phages employ the arbitrium communication system, which contains an arbitrium hexapeptide, the cellular receptor AimR and the lysogenic negative regulator AimX. This system controls the decision between the lytic and lysogenic cycles. However, both the mechanism of molecular recognition between the arbitrium peptide and AimR and how downstream gene expression is regulated remain unknown. Here, we report crystal structures for AimR from the SPbeta phage in the apo form and the arbitrium peptide-bound form at 2.20 Å and 1.92 Å, respectively. With or without the peptide, AimR dimerizes through the C-terminal capping helix. AimR assembles a superhelical fold and accommodates the peptide encircled by its tetratricopeptide repeats, which is reminiscent of RRNPP family members from the quorum-sensing system. In the absence of the arbitrium peptide, AimR targets the upstream sequence of the aimX gene; its DNA binding activity is prevented following peptide binding. In summary, our findings provide a structural basis for peptide recognition in the phage lysis-lysogeny decision communication system.

A severe mouse model of spinal muscular atrophy develops early systemic inflammation.

Spinal muscular atrophy (SMA) is a fatal genetic disease, mainly affecting children. A number of recent studies show, aside from lower motor neuron degeneration and atrophy of skeletal muscles, widespread defects present in the central nervous system (CNS) and peripheral non-neuronal cell types of SMA patients and mouse models, particularly of severe forms. However, molecular mechanisms underlying the multi-organ manifestations of SMA were hardly understood. Here, using histology, flow cytometry and gene expression analysis in both messenger RNA and protein levels in various tissues, we found that a severe SMA mouse model develops systemic inflammation in early symptomatic stages. SMA mice had an enhanced intestinal permeability, resulting in microbial invasion into the circulatory system. Expression of proinflammatory cytokines was increased in all tissues and the acute phase response in the liver was activated. Systemic inflammation further mobilized glucocorticoid signaling and in turn led to dysregulation of a large set of genes, including robust upregulation of FAM107A in the spinal cord, increased expression of which has been implicated in neurodegeneration. Moreover, we show that lipopolysaccharide challenge markedly suppressed survival of motor neuron 2 exon 7 splicing in all examined peripheral and CNS tissues, resulting in global survival of motor neuron level reduction. Therefore, we identified a novel pathological mechanism in a severe SMA mouse model, which affects phenotypic severity through multiple paths and should contribute to progression of broad neuronal and non-neuronal defects.

Structural Insights into the Inhibition of Tubulin by the Antitumor Agent 4ß-(1,2,4-triazol-3-ylthio)-4-deoxypodophyllotoxin.

The colchicine domain is widely recognized as the binding site of microtubule depolymerization agents for anticancer drug design. Almost all of the drugs targeting the colchicine domain have been confirmed to bind to the tubulin ß-subunit. Here we studied a crystal structure (2.3 Å) of the complex between tubulin and 4ß-(1,2,4-triazol-3-ylthio)-4-deoxypodophyllotoxin (compound 1S) with superior antitumor activity, which was designed on the basis of the colchicine domain and synthesized in our previous work. A distinct binding model of the colchicine domain was found in the complexes of tubulin with compound 1S. From a comparison of the crystal structures of tubulin-compound 1S and tubulin-colchicine complexes, the side chains of the T7 loop of ß-tubulin flip outward and the T5 loop of α-tubulin changes its conformation. It has been shown that the ß-subunit T7 loop reversibly participates in resistance to straightening that opposes microtubule assembly by flipping in and out. Together with the biochemical results from compound 1S, the structural data highlight the main contributors in the α-subunits and the colchicine domain ß-subunits: the dual-target binding sites in the α-T7 loop and ß-H7-T7 loop of tubulin. Compound 1S can synchronously bind to αß-tubulin. The structures also highlight common features for the design and development of novel potent microtubule destabilizing agents.

Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.

Chemical modifications of RNA have essential roles in a vast range of cellular processes. N(6)-methyladenosine (m(6)A) is an abundant internal modification in messenger RNA and long non-coding RNA that can be dynamically added and removed by RNA methyltransferases (MTases) and demethylases, respectively. An MTase complex comprising methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) efficiently catalyses methyl group transfer. In contrast to the well-studied DNA MTase, the exact roles of these two RNA MTases in the complex remain to be elucidated. Here we report the crystal structures of the METTL3-METTL14 heterodimer with MTase domains in the ligand-free, S-adenosyl methionine (AdoMet)-bound and S-adenosyl homocysteine (AdoHcy)-bound states, with resolutions of 1.9, 1.71 and 1.61 Å, respectively. Both METTL3 and METTL14 adopt a class I MTase fold and they interact with each other via an extensive hydrogen bonding network, generating a positively charged groove. Notably, AdoMet was observed in only the METTL3 pocket and not in METTL14. Combined with biochemical analysis, these results suggest that in the m(6)A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N(6)-adenine MTase. This structural information provides an important framework for the functional investigation of m(6)A.