Cappello Anna R, Miniero Daniela V, Curcio Rosita, Ludovico Anna, Daddabbo Lucia, Stipani Italo, Robinson Alan J, Kunji Edmund R S, Palmieri Ferdinando
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy.
J Mol Biol. 2007 Jun 1;369(2):400-12. doi: 10.1016/j.jmb.2007.03.048. Epub 2007 Mar 24.
The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents. The observed effects were rationalized by using a comparative structural model of the OGC. Most of the residues that are critical for function are found at the bottom of the cavity and they belong to the signature motifs P-X-[DE]-X-X-[KR] that form a network of three inter-helical salt bridges that close the carrier at the matrix side. The OGC deviates from most other carriers, because it has a conserved leucine (L144) rather than a positively charged residue in the signature motif of the second repeat and thus the salt bridge network is lacking one salt bridge. Incomplete salt-bridge networks due to hydrophobic, aromatic or polar substitutions are observed in other dicarboxylate, phosphate and adenine nucleotide transporters. The interaction between the carrier and the substrate has to provide the activation energy to trigger the re-arrangement of the salt-bridge network and other structural changes required for substrate translocation. For substrates such as malate, which has only two carboxylic and one hydroxyl group, a reduction in the number of salt bridges in the network may be required to lower the energy barrier for translocation. Another group of key residues, consisting of T36, A134, and T233, is close to the putative substrate binding site and substitutions or modifications of these residues may interfere with substrate binding and ion coupling. Residues G32, A35, Q40, G130, G133, A134, G230, and S237 are potentially engaged in inter-helical interactions and they may be involved in the movements of the alpha-helices during translocation.
线粒体α-酮戊二酸载体（OGC）在苹果酸-天冬氨酸穿梭、α-酮戊二酸-异柠檬酸穿梭以及糖异生过程中发挥着重要作用。为确定对功能至关重要的氨基酸残基，在一个完全功能性且不含半胱氨酸残基的突变载体中，将跨膜α-螺旋H1、H3和H5中的每个残基都系统地替换为半胱氨酸。在有和没有巯基试剂存在的情况下测量突变载体的转运活性。通过使用OGC的比较结构模型对观察到的效应进行了合理解释。大多数对功能至关重要的残基位于腔的底部，它们属于特征基序P-X-[DE]-X-X-[KR]，这些基序形成了三个螺旋间盐桥网络，在基质侧封闭载体。OGC与大多数其他载体不同，因为它在第二个重复序列的特征基序中有一个保守的亮氨酸（L144）而不是带正电荷的残基，因此盐桥网络缺少一个盐桥。在其他二羧酸、磷酸盐和腺嘌呤核苷酸转运体中也观察到由于疏水、芳香或极性取代导致的不完全盐桥网络。载体与底物之间的相互作用必须提供活化能，以触发盐桥网络的重新排列以及底物转运所需的其他结构变化。对于像苹果酸这样只有两个羧基和一个羟基的底物，可能需要减少网络中盐桥的数量以降低转运的能量屏障。另一组关键残基，由T36、A134和T233组成，靠近假定的底物结合位点，这些残基的取代或修饰可能会干扰底物结合和离子偶联。残基G32、A35、Q40、G130、G133、A134、G230和S237可能参与螺旋间相互作用，并且它们可能在转运过程中参与α-螺旋的运动。
