Mitochondrial pyruvate carrier (MPC) is a mitochondrial inner membrane protein complex essential for uptake of pyruvate into matrix as the primary carbon source for tricarboxylic acid (TCA) cycle^1,2. Here, we report six cryo-EM structures of human MPC in three different states: three structures obtained at different conditions in intermembrane space (IMS)-open state with highest resolution of 3.2 Å, a structure of pyruvate-treated MPC in occluded state at 3.7 Å, and two structures in matrix-facing state bound with the inhibitor UK5099 or an inhibitory nanobody on the matrix side at 3.2 Å and 3.0 Å, respectively. MPC is assigned into a heterodimer consisting of MPC1 and MPC2, with the transmembrane domain adopting pseudo-C2-symmetry. Approximate rigid body movements occur between the IMS-open state and the occluded state, while structural changes primarily on the matrix side facilitate the transition between the occluded state and the matrix-facing state, revealing the alternating access mechanism during pyruvate transport. In the UK5099-bound structure, the inhibitor fits well and interacts extensively with a pocket that opens to the matrix side. Our findings provide important insights into the mechanisms underlying MPC-mediated substrate transport, and the recognition and inhibition by UK5099, which will facilitate future drug development targeting MPC.

线粒体丙酮酸载体 （MPC） 是一种线粒体内膜蛋白复合物，对于丙酮酸作为三羧酸 （TCA） 循环的主要碳源^1,2 将丙酮酸摄取到基质中至关重要。在这里，我们报道了三种不同状态下人类 MPC 的六种冷冻电镜结构：在不同条件下以膜间隙 （IMS） 开放状态获得的三种结构，最高分辨率为 3.2 Å，一种丙酮酸处理的 MPC 在 3.7 Å 的闭塞状态下的结构，以及两种面向基质的状态下与抑制剂 UK5099 结合的结构或在 3.2 Å 和 3.0 Å 的基质侧的抑制性纳米抗体， 分别。MPC 被分配到由 MPC1 和 MPC2 组成的异二聚体中，跨膜结构域采用伪 C2 对称性。近似的刚体运动发生在 IMS 开放状态和闭塞状态之间，而主要在基体侧的结构变化促进了闭塞状态和面向基体状态之间的过渡，揭示了丙酮酸运输过程中的交替进入机制。在 UK5099 结合的结构中，抑制剂拟合良好，并与向基质侧打开的口袋广泛相互作用。我们的研究结果为 MPC 介导的底物转运机制以及 UK5099 的识别和抑制提供了重要见解，这将促进未来针对 MPC 的药物开发。