Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0–3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.

质子偶联的单羧酸转运蛋白 MCT1-4 催化代谢必需的单羧酸的跨膜运动，并因其在各种肿瘤中的表达增强而成为癌症治疗的目标。在这里，我们报告了五种冷冻 EM 结构，分辨率为 3.0-3.3 Å，在 Basigin-2 存在的情况下，人类 MCT1 与乳酸或抑制剂结合，Basigin-2 是一种含有单跨膜片段 (TM) 的伴侣。当与乳酸或抑制剂 BAY-8002 和 AZD3965 复合时，MCT1 表现出类似的向外开放构象。在抑制剂 7ACC2 存在或 Asn 中和质子偶联残基 Asp309 的情况下，捕获了类似的向内开放结构。辅以结构引导的生化分析，我们的研究揭示了 MCT 的底物结合和转运机制，阐明三种抗癌候选药物的作用模式，并确定 MCT1 和 MCT4 对 AZD3965 亚型特异性敏感性的决定因素。这些发现为靶向 MCT 的结构引导药物发现奠定了重要的框架。