Washington Erica J, Zhou Ye, Hsu Allen L, Petrovich Matthew, Tenor Jennifer L, Toffaletti Dena L, Guan Ziqiang, Perfect John R, Borgnia Mario J, Bartesaghi Alberto, Brennan Richard G
bioRxiv. 2024 Jun 5:2023.03.14.530545. doi: 10.1101/2023.03.14.530545.
Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies residues required for substrate-binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. , these studies expand our knowledge of trehalose biosynthesis in and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes.
Fungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to transition from the environment to the human host. Enzymes in the trehalose biosynthesis pathway are absent in humans and, therefore, are potentially significant targets for novel antifungal therapeutics. One enzyme in the trehalose biosynthesis is trehalose-6-phosphate synthase (Tps1). Here, we describe the cryo-electron microscopy structures of the CnTps1 homo-tetramer in the unliganded form and in complex with a substrate and a product. These structures and subsequent biochemical analysis reveal key details of substrate-binding residues and substrate specificity. These structures should facilitate structure-guided design of inhibitors against CnTps1.
侵袭性真菌疾病是对人类健康的重大威胁，在全球每年导致超过150万人死亡。抗真菌治疗药物的种类仍然有限，迫切需要针对人类所没有的其他生物合成途径的新型药物。其中一条途径涉及海藻糖的生物合成。海藻糖是一种二糖，致病真菌在人类宿主中生存需要它。在海藻糖生物合成的第一步中，海藻糖-6-磷酸合酶（Tps1）将尿苷二磷酸葡萄糖和6-磷酸葡萄糖转化为海藻糖-6-磷酸。在此，我们报道了全长Tps1（CnTps1）未结合配体形式以及与尿苷二磷酸和6-磷酸葡萄糖形成复合物的结构。对这两种结构的比较揭示了配体结合后N端向催化口袋的显著移动，并确定了底物结合所需的残基以及稳定四聚体的残基。有趣的是，一个内在无序结构域（IDD），在隐球菌属物种和密切相关的担子菌中保守，从四聚体的每个亚基延伸到“溶剂”中，但在密度图中不可见。我们确定IDD对于Tps1依赖的耐热性和渗透压应激生存不是必需的。用尿苷二磷酸半乳糖进行的研究突出了CnTps1精确的底物特异性。这些研究扩展了我们对隐球菌中海藻糖生物合成的认识，并突出了开发破坏这种二糖合成或功能性四聚体形成的抗真菌治疗药物的潜力，以及冷冻电镜在CnTps1-配体/药物复合物结构表征中的应用。
真菌感染每年在全球导致超过一百万人死亡。多种致病真菌从环境过渡到人类宿主需要二糖海藻糖的生物合成。海藻糖生物合成途径中的酶在人类中不存在，因此是新型抗真菌治疗药物的潜在重要靶点。海藻糖生物合成中的一种酶是海藻糖-6-磷酸合酶（Tps1）。在此，我们描述了未结合配体形式以及与一种底物和一种产物形成复合物的CnTps1同型四聚体的冷冻电镜结构。这些结构以及随后的生化分析揭示了底物结合残基和底物特异性的关键细节。这些结构应有助于针对CnTps1的抑制剂的结构导向设计。
