Structural insights into the Oryza sativa cation transporters HKTs in salt tolerance.

The high-affinity potassium transporters (HKTs), selectively permeable to either Na+ alone or Na+/K+, play pivotal roles in maintaining plant Na+/K+ homeostasis. Although their involvement in salt tolerance is widely reported, the molecular underpinnings of Oryza sativa HKTs remain elusive. In this study, we elucidate the structures of OsHKT1;1 and OsHKT2;1, representing two distinct classes of rice HKTs. The dimeric assembled OsHKTs can be structurally divided into four domains. At the dimer interface, a half-helix or a loop in the third domain is coordinated by the C-terminal region of the opposite subunit. Additionally, we present the structures of OsHKT1;5 salt-tolerant and salt-sensitive variants, a key quantitative trait locus associated with salt tolerance. The salt-tolerant variant of OsHKT1;5 exhibits enhanced Na+ transport capability and displays a more flexible conformation. These findings shed light on the molecular basis of rice HKTs and provide insights into their role in salt tolerance.

Structural basis for the activity regulation of Salt Overly Sensitive 1 in Arabidopsis salt tolerance.

The plasma membrane Na+/H+ exchanger Salt Overly Sensitive 1 (SOS1) is crucial for plant salt tolerance. Unlike typical sodium/proton exchangers, SOS1 contains a large cytoplasmic domain (CPD) that regulates Na+/H+ exchange activity. However, the underlying modulation mechanism remains unclear. Here we report the structures of SOS1 from Arabidopsis thaliana in two conformations, primarily differing in CPD flexibility. The CPD comprises an interfacial domain, a cyclic nucleotide-binding domain-like domain (CNBD-like domain) and an autoinhibition domain. Through yeast cell-based Na+ tolerance test, we reveal the regulatory role of the interfacial domain and the activation role of the CNBD-like domain. The CPD forms a negatively charged cavity that is connected to the ion binding site. The transport of Na+ may be coupled with the conformational change of CPD. These findings provide structural and functional insight into SOS1 activity regulation.

Macrophage scavenger receptor A1 antagonizes abdominal aortic aneurysm via upregulating IRG1.

AIMS: Abdominal aortic aneurysm (AAA) is a common, usually asymptomatic disease with high mortality and limited therapeutic options. Extensive extracellular matrix (ECM) fragmentation and transmural inflammation act as major pathological processes of AAA. However, the underlying regulatory mechanisms remain incompletely understood. Herein, we aimed to investigate the role of scavenger receptor A1 (SR-A1), a key pattern recognition receptor modulating macrophage activity, in pathogenesis of AAA. METHODS AND RESULTS: The AAA model was generated by administration of angiotensin II (Ang II) into apolipoprotein E knockout mice or peri-arterial application of calcium phosphate in C57BJ/6L mice. We found that SR-A1 was markedly down-regulated in the macrophages isolated from murine AAA aortas. Global or myeloid-specific ablation of SR-A1 aggravated vascular inflammation, loss of vascular smooth muscle cells and degradation of the extracellular matrix. These effects of SR-A1 deficiency on AAA development were mediated by suppressed immunoresponsive gene 1 (IRG1) and increased inflammatory response in macrophages. Mechanically, binding of SR-A1 with Lyn led to STAT3 phosphorylation and translocation into the nucleus, in which STAT3 promoted IRG1 transcription through directly binding to its promoter. Restoration of macrophage SR-A1 in SR-A1-deficient mice by bone marrow transplantation or administration of 4-octyl itaconate, the derivate of IRG1 product itaconate, could relieve murine AAA. CONCLUSION: Our study reveals a protective effect of macrophage SR-A1-STAT3-IRG1 axis against aortic aneurysm formation via inhibiting inflammation.

Structural and functional insights of the human peroxisomal ABC transporter ALDP.

Adrenoleukodystrophy protein (ALDP) is responsible for the transport of very-long-chain fatty acids (VLCFAs) and corresponding CoA-esters across the peroxisomal membrane. Dysfunction of ALDP leads to peroxisomal metabolic disorder exemplified by X-linked adrenoleukodystrophy (ALD). Hundreds of ALD-causing mutations have been identified on ALDP. However, the pathogenic mechanisms of these mutations are restricted to clinical description due to limited structural and biochemical characterization. Here we report the cryo-electron microscopy structure of human ALDP with nominal resolution at 3.4 Å. ALDP exhibits a cytosolic-facing conformation. Compared to other lipid ATP-binding cassette transporters, ALDP has two substrate binding cavities formed by the transmembrane domains. Such structural organization may be suitable for the coordination of VLCFAs. Based on the structure, we performed integrative analysis of the cellular trafficking, protein thermostability, ATP hydrolysis, and the transport activity of representative mutations. These results provide a framework for understanding the working mechanism of ALDP and pathogenic roles of disease-associated mutations.

Structural basis for the activity regulation of a potassium channel AKT1 from Arabidopsis.

The voltage-gated potassium channel AKT1 is responsible for primary K+ uptake in Arabidopsis roots. AKT1 is functionally activated through phosphorylation and negatively regulated by a potassium channel α-subunit AtKC1. However, the molecular basis for the modulation mechanism remains unclear. Here we report the structures of AKT1, phosphorylated-AKT1, a constitutively-active variant, and AKT1-AtKC1 complex. AKT1 is assembled in 2-fold symmetry at the cytoplasmic domain. Such organization appears to sterically hinder the reorientation of C-linkers during ion permeation. Phosphorylated-AKT1 adopts an alternate 4-fold symmetric conformation at cytoplasmic domain, which indicates conformational changes associated with symmetry switch during channel activation. To corroborate this finding, we perform structure-guided mutagenesis to disrupt the dimeric interface and identify a constitutively-active variant Asp379Ala mediates K+ permeation independently of phosphorylation. This variant predominantly adopts a 4-fold symmetric conformation. Furthermore, the AKT1-AtKC1 complex assembles in 2-fold symmetry. Together, our work reveals structural insight into the regulatory mechanism for AKT1.

Identification of four ene reductases and their preliminary exploration in the asymmetric synthesis of (R)-dihydrocarvone and (R)-profen derivatives.

The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens, Pseudomonas fluorescens, and Pseudomonas veronil. The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress.