
GABA轉運體(GAT1)利用鈉離子的結合來穩定其構象,促進GABA的運輸。鈉離子的招募和結合位點在調節GAT1的功能中扮演關鍵角色。


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The human GABA transporter (GAT1) is a membrane transporter that mediates the reuptake of the neurotransmitter GABA from the synaptic cleft into neurons and glial cells. Dysregulation of the transport cycle has been associated with epilepsy and neuropsychiatric disorders, highlighting the crucial role of the transporter in maintaining homeostasis of brain GABA levels. GAT1 is a secondary active transporter that couples the movement of substrate to the simultaneous transport of sodium and chloride ions along their electrochemical gradients. Using MD simulations, we identified a novel sodium recruiting site at the entrance to the outer vestibule, which attracts positively charged ions and increases the local sodium concentration, thereby indirectly increasing sodium affinity. Mutations of negatively charged residues at the recruiting site slowed the binding kinetics, while experimental data revealed a change in sodium dependency of GABA uptake and a reduction of sodium affinity. Simulation showed that sodium displays a higher affinity for the sodium binding site NA2, which plays a role in stabilisation of the outward-open conformation. We directly show that the presence of a sodium ion bound to NA2 increases the stability of the closed inner gate and restrains motions of TM5. We find that sodium is only weakly bound to NA1 in the absence of GABA, while the presence of the substrate strengthens the interaction due to the completed ion coordinating shell, explaining cooperativity between GABA and sodium.

### Competing Interest Statement

The authors have declared no competing interest.

Interaction of GAT1 with sodium ions: from efficient recruitment to stabilisation of substrate and conformation

人類gaba轉運體-gat1-與鈉離子的相互作用-從有效招募到基質和構象的穩定


人類GABA轉運體(GAT1)與鈉離子的相互作用:從有效招募到基質和構象的穩定


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生物大分子如蛋白質、DNA/RNA及其複合物的結構動力學受到其許多可滴定基團的質子化變化的強烈影響,解釋了它們對pH變化的敏感性。相反地,生物大分子的構象和環境變化也會影響這些基團的質子化狀態。我們開發了一種基於GPU加速的快速多極法(FMM)電磁力學的嚴格的哈密頓插值λ動力學恆定pH方法,支持CHARMM36m和Amber99sb*-ILDN力場,並實現了自動化,使從常規MD到恆定pH MD的切換變得輕鬆。


以快速多極法電磁力學在gromacs中進行恆定ph模擬-設計與應用


以快速多極法電磁力學在GROMACS中進行恆定pH模擬：設計與應用



哺乳動物膜結合腺苷酸環化酶的膜錨定區域具有受體功能,可以被特定脂質配體調控,從而調節Gsα刺激下的cAMP生成。


The biosynthesis of cAMP by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled-receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1 to 7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo . Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5- mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR-Gsα signaling regulating the biosynthesis of cAMP.

### Competing Interest Statement

The authors have declared no competing interest.

A new class of receptors: Lipids regulate mammalian Gsα-stimulated adenylyl cyclase activities via their membrane anchors

哺乳動物gsα刺激的腺苷酸環化酶活性受脂質膜錨定調控的新受體類型


哺乳動物Gsα刺激的腺苷酸環化酶活性受脂質膜錨定調控的新受體類型



水和氯化物可以通過結合到WNK激酶的非活性二聚體構型來抑制其活性。


Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1and CWN2). Here we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride binding site and are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 Cluster mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.

### Competing Interest Statement

The authors have declared no competing interest.

Water and chloride as allosteric inhibitors in WNK kinase osmosensing

水和氯化物作為wnk激酶滲透感知的致敏抑制劑


水和氯化物作為WNK激酶滲透感知的致敏抑制劑



脂質單層膜中Na+和K+離子比例的變化會導致膜的剛性、分子角度和鍵結性質發生非線性變化,表明界面存在空間變化的電場。


水中na-k比對脂質-水界面的空間變化電場


水中Na/K比對脂質-水界面的空間變化電場



細胞膜的高滲透性會壓倒任何由巨分子凝聚提供的滲透壓緩衝能力。


[Watson et al . (2023][1], Macromolecular condensation buffers intracellular water potential, Nature 623: 842-852) have proposed that the reversible formation and disassembly of molecular condensates could act as the primary buffer of cytoplasmic osmolality in the face of changes in extracellular osmolality. In this communication, I show using well-established membrane biophysics, that the water permeability of plasma membranes is likely to overwhelm any cytoplasmic water buffers.

### Competing Interest Statement

The authors have declared no competing interest.

 [1]: #ref-27

Macromolecular condensation is unlikely to buffer intracellular osmolality

細胞內滲透壓調節不太可能依賴巨分子凝聚


乳酸脫氫酶的抑制通過動力學和熱力學機制調控糖酵解、三羧酸循環和氧化磷酸化,從而影響癌細胞的能量代謝。


Lactate dehydrogenase (LDH) stands at the intersection of pyruvate metabolism. While it is believed that inhibition of LDH redirects pyruvate to mitochondrial metabolism, suppressing glycolysis and boosting oxidative phosphorylation, the mechanism remains largely unexplored. We found that individual LDH A or B knockouts had minimal impact on glycolysis, tricarboxylic acid cycle (TCAC), or oxidative phosphorylation (OXPHOS). However, combining LDH knockout with LDH inhibitor GNE-140 significantly suppressed these processes. Inhibition of LDH led to an increase in free NADH concentration and a decrease in free NAD+ concentration, the reduced free NAD+ concentration inhibited GAPDH, disrupting the balance of glycolytic intermediates, which were linked with thermodynamic shift of the Gibbs free energy of reactions between phosphofructokinase 1 (PFK1) and phosphoglycerate mutase (PGAM) in the glycolytic pathway, favoring their reverse direction. This disrupted glycolysis led to impaired TCAC and mitochondrial respiration due to reduced pyruvate and glutamine carbon influx into TCAC. Under hypoxia, LDH inhibition had a stronger effect, inducing energy crisis, redox imbalance, and cancer cell death. Our study reveals LDH’s intricate control over glycolysis, TCAC, and mitochondrial respiration, highlighting the interplay of enzyme kinetics and thermodynamics in metabolic pathways—a crucial aspect for understanding metabolic regulation.

Impact statement The biochemical mechanism underlying the flux regulation of glycolytic flux by lactate dehydrogenase in cancer cells, which have remained largely unexplored, is deciphered.

Funding information This work has been supported in part by China Natural Science Foundation projects (82073038, 81772947), a key project (2018C03009) funded by Zhejiang Provincial Department of Sciences and Technologies, the Fundamental Research Funds for the Central Universities (226-2024-00062), to X.H.

### Competing Interest Statement

The authors have declared no competing interest.

Elucidating the kinetic and thermodynamic insight into regulation of glycolysis by lactate dehydrogenase and its impact on tricarboxylic acid cycle and oxidative phosphorylation in cancer cells

探討乳酸脫氫酶對癌細胞糖酵解-三羧酸循環和氧化磷酸化的調控機制及其影響


探討乳酸脫氫酶對癌細胞糖酵解、三羧酸循環和氧化磷酸化的調控機制及其影響



建立了從過飽和度測量計算蛋白質溶液滲透第二維里係數的方法,並將其與小角X射線散射法得到的結果進行比較。


蛋白質溶液的滲透第二維里係數的替代方法及其在液-液相分離中的應用


組蛋白可離子化殘基的質子化狀態變化會顯著影響核小體表面的靜電勢和核小體與調控蛋白的相互作用。


The nucleosome serves as the fundamental unit of chromatin organization, with electrostatic interactions acting as the driving forces in the folding of nucleosomes into chromatin. Perturbations in cellular pH conditions can lead to changes in the protonation states of titratable histone residues, impacting nucleosome surface electrostatic potentials and interactions. However, the effects of proton uptake or release of histone ionizable groups on nucleosome-partner protein interactions and higher-order chromatin structures remain largely unexplored. Here, we conducted comprehensive analyses of histone titratable residue pKa values in various nucleosome contexts, utilizing 96 experimentally determined structures. We revealed that pH-induced changes in histone residue protonation states modulated nucleosome surface electrostatic potentials and significantly influenced nucleosome-partner protein interactions. Furthermore, we observed that proton uptake or release often accompanied nucleosome-partner protein interactions, facilitating their binding processes. Additionally, using a dataset of 1266 recurrent histone cancer mutations, we systematically characterized their impact on nucleosome surface electrostatics, demonstrating their profound effects on electrostatic interactions between nucleosomes and partner proteins. Finally, our findings suggest that alterations in histone protonation or cancer mutations can also regulate nucleosome self-association, thereby modulating the organization and dynamics of higher-order chromatin structure.

### Competing Interest Statement

The authors have declared no competing interest.

Electrostatic interactions in nucleosome and higher-order structures are regulated by protonation state of histone ionizable residue

核小體和高階結構中的靜電相互作用受到組蛋白可離子化殘基質子化狀態的調節


利用工程化的醛縮酶活性位點中產生的亞胺離子中間體吸收紫光,可以活化手性羧酸,經脫羧產生兩個自由基,進而發生立體專一性的自由基偶合反應,合成具有兩個手性中心的產物。


Stereospecific radical coupling with a non-natural photodecarboxylase - Nature

利用非天然光脫羧酶進行立體專一性自由基偶合反應