Yuxing Chen

In plants, peroxisomal β oxidation is essential for energy production, sugar metabolism and the regulation of hormonal signaling. The Arabidopsis COMATOSE (CTS) gene, which encodes the peroxisomal ATP-binding cassette transporter ABCD1 (also known as CTS or PAX1), mediates the import of various fatty acyls and hormones into peroxisomes for β oxidation. Although physiological evidence has demonstrated that defects in CTS impair plant development and growth, the underlying molecular mechanisms remain poorly understood. In this study, biochemical assays confirm that CTS transports a broad range of fatty acyl-CoAs, as well as indole-3-butyric acid (IBA), an auxin and precursor to indole-3-aceditc acid, and auxin herbicide 2,4-dichlorophenoxy butyric acid (2,4-DB). We further solve five cryo-EM structures of CTS: the apo form, ATP-bound, and three substrate-bound states (C12:0-, IBA- and 2,4-DB-CoA) at near-atom resolutions. These structures reveal that CTS binds a single substrate molecule in a characteristic U-shaped conformation. Moreover, physiological studies using point mutations of key substrate-binding residues identify critical residues essential for its function. Our findings reveal the molecular principles by which CTS integrates lipid metabolism, hormone regulation, and xenobiotic activation throughout the plant life cycle, as well as underscore its potential as a target for crop improvement and herbicide development.

Maintaining carbon/nitrogen (C/N) metabolic balance is essential for cellular homeostasis, allowing microorganisms to adapt to fluctuating environmental conditions. In the autotrophic cyanobacteria, the C/N balance is achieved through a sophisticated network that coordinates the uptake of inorganic carbon and nitrogen, including the ATP-binding cassette (ABC) transporters CmpABCD and NRT that import bicarbonate and nitrate, respectively. Notably, both transporters possess an extra C-terminal regulatory domain (CRD) that is fused to one of the nucleotide-binding domains (NBDs). Via structure guided site-directed mutagenesis and bicarbonate transport activity assays, we found that CmpABCD is tightly regulated by the nitrate-binding CRD. At a low intracellular nitrate concentration, CmpBCD adopts an auto-inhibited conformation, in which the CRD locks the two NBDs of CmpC and CmpD. Upon binding to the nitrate, the CRD is released from NBDs and becomes highly flexible, thus restoring the transport activity of CmpABCD. We propose a distinct regulatory mechanism of ABC transporters, which may be broadly applicable to those fused with a regulatory domain. Moreover, these findings combined with previous reports establish a direct link between the inorganic carbon uptake and intracellular nitrate level through an ABC importer, providing a straightforward and economic strategy that coordinates the C/N homeostasis.

OSTα/β is the essential basolateral transporter responsible for returning reabsorbed bile acids to the portal circulation, yet its molecular mechanism remains unclear. Here, we report the cryo-electron microscopy structure of human OSTα/β at 2.7 Å resolution, revealing a GPCR-like 7-TM architecture distinct from all established SLC transporter folds. We further determined the taurocholate (TC)-bound structure at 3.2 Å, showing that the substrate laterally binds to a surface groove on OSTα, but not in the central cavity. The TC molecule is captured by a line of conserved polar residues along the groove, which were further proved by structure-guided mutagenesis combined with transport assays. Molecular dynamics simulations also suggest that it is energetically favored for TC moving along the surface groove. Together, structural analysis, functional assays, and simulations demonstrate that OSTα/β mediates bile acid transmembrane translocation via a facilitated lateral diffusion mechanism with subtle conformational change, which is distinct from known SLCs. These findings establish a mechanistic framework for OSTα/β function in enterohepatic bile acid circulation and provide a foundation for therapeutic modulation of bile acid homeostasis.

Summary: Carboxysomes are self‐assembled bacterial microcompartments (BMCs) that encapsulate the enzymes RuBisCO and carbonic anhydrase into a proteinaceous shell, enhancing the efficiency of photosynthetic carbon fixation. The chaperone CcmS was reported to participate in the assembly of β‐carboxysomes; however, the underlying molecular mechanism remains elusive. We report the crystal structure of CcmS from Synechocystis sp. PCC 6803, revealing a monomer of α/β fold. Moreover, its complex structures with two types of BMC hexamers, CcmK1 homohexamer and CcmK1‐CcmK2 heterohexamer, reveal a same pattern of CcmS binding to the featured C‐terminal segment of CcmK1. Upon binding to CcmS, this C‐terminal segment of CcmK1 is folded into an amphipathic α‐helix protruding outward that might function as a hinge to crosslink adjacent BMC‐H hexamers, thereby facilitating concerted and precise assembly of the β‐carboxysome shell. Deletion of the ccmS gene or the 8‐residue C‐terminal coding region of ccmK1 resulted in the formation of aberrant and fewer carboxysomes, suppressed photosynthetic capacity in Synechocystis sp. PCC 6803. These findings enable us to propose a putative model for the chaperone‐assisted assembly of β‐carboxysome shell and provide clues for the design and engineering of efficient carbon fixation machinery.

Recent language models have significantly accelerated our understanding on the massive biological data, using protein or DNA/RNA sequences as a single-language modality. Here we present a dual-language foundation model, which integrates both protein and coding sequences (CDS) for pre-training. Compared to the benchmark models, it shows a superior performance up to ∼20% on both protein and mRNA-related discriminative tasks, and gains the capacity to de novo generate coding sequences of ∼50% increased protein yield. Moreover, the model also possesses the knowledge transferability from the pre-training data to the upstream 5’ untranslated regions. These findings indicate the intrinsic correlations between protein and its CDS, as well as the coding region and beyond. It provides a new paradigm that leverages the multiple-language foundation model to interpret the hidden context of distinct corpora/biological languages, which could be further applied to mine the yet-unknown biological information/correlation beyond the Central Dogma.

Human ABC transporters ABCD1–3 are all localized on the peroxisomal membrane and participate in the β-oxidation of fatty acyl-CoAs, but they differ from each other in substrate specificity. The transport of branched-chain fatty acids from cytosol to peroxisome is specifically driven by ABCD3, dysfunction of which causes severe liver diseases such as hepatosplenomegaly. Here we report two cryogenic electron microscopy (cryo-EM) structures of ABCD3 bound to phytanoyl-CoA and ATP at resolutions of 2.9 Å and 3.2 Å, respectively. A pair of phytanoyl-CoA molecules were observed in ABCD3, each binding to one transmembrane domain (TMD), which is distinct from our previously reported structure of ABCD1, where each fatty acyl-CoA molecule strongly crosslinks two TMDs. Upon ATP binding, ABCD3 exhibits a conformation that is open towards the peroxisomal matrix, leaving two extra densities corresponding to two CoA molecules deeply embedded in the translocation cavity. Structural analysis combined with substrate-stimulated ATPase activity assays indicated that the present structures might represent two states of ABCD3 in the transport cycle. These findings advance our understanding of fatty acid oxidation and the molecular pathology of related diseases.

Carboxysomes are bacterial microcompartments that encapsulate the enzymes RuBisCO and carbonic anhydrase in a proteinaceous shell to enhance the efficiency of photosynthetic carbon fixation. The self-assembly principles of the intact carboxysome remain elusive. Here we purified α-carboxysomes from Prochlorococcus and examined their intact structures using single-particle cryo-electron microscopy to solve the basic principles of their shell construction and internal RuBisCO organization. The 4.2 Å icosahedral-like shell structure reveals 24 CsoS1 hexamers on each facet and one CsoS4A pentamer at each vertex. RuBisCOs are organized into three concentric layers within the shell, consisting of 72, 32 and up to 4 RuBisCOs at the outer, middle and inner layers, respectively. We uniquely show how full-length and shorter forms of the scaffolding protein CsoS2 bind to the inner surface of the shell via repetitive motifs in the middle and C-terminal regions. Combined with previous reports, we propose a concomitant ‘outside-in’ assembly principle of α-carboxysomes: the inner surface of the self-assembled shell is reinforced by the middle and C-terminal motifs of the scaffolding protein, while the free N-terminal motifs cluster to recruit RuBisCO in concentric, three-layered spherical arrangements. These new insights into the coordinated assembly of α-carboxysomes may guide the rational design and repurposing of carboxysome structures for improving plant photosynthetic efficiency.

The Myoviridae cyanophage A-1(L) specifically infects the model cyanobacteria Anabaena sp. PCC 7120. Following our recent report on the capsid structure of A-1(L), here we present the high-resolution cryo-EM structure of its intact tail machine including the neck, tail and attached fibers. Besides the dodecameric portal, the neck contains a canonical hexamer connected to a unique pentadecamer that anchors five extended bead-chain-like neck fibers. The 1045-Å-long contractile tail is composed of a helical bundle of tape measure proteins surrounded by a layer of tube proteins and a layer of sheath proteins, ended with a five-component baseplate. The six long and six short tail fibers are folded back pairwise, each with one end anchoring to the baseplate and the distal end pointing to the capsid. Structural analysis combined with biochemical assays further enable us to identify the dual hydrolytic activities of the baseplate hub, in addition to two host receptor binding domains in the tail fibers. Moreover, the structure of the intact A-1(L) also helps us to reannotate its genome. These findings will facilitate the application of A-1(L) as a chassis cyanophage in synthetic biology.