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牛线粒体氧代戊二酸载体偶数跨膜α螺旋中氨基酸残基的功能和结构作用

Functional and structural role of amino acid residues in the even-numbered transmembrane alpha-helices of the bovine mitochondrial oxoglutarate carrier.

作者信息

Cappello Anna Rita, Curcio Rosita, Valeria Miniero Daniela, Stipani Italo, Robinson Alan J, Kunji Edmund R S, Palmieri Ferdinando

机构信息

Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy.

出版信息

J Mol Biol. 2006 Oct 13;363(1):51-62. doi: 10.1016/j.jmb.2006.08.041. Epub 2006 Aug 22.

Abstract

The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. Orthologs of the carrier have a high degree of amino acid sequence conservation, meaning that it is impossible to identify residues important for function on the basis of this criterion alone. Therefore, each amino acid residue in the transmembrane alpha-helices H2 and H6 was replaced by a cysteine in a functional mitochondrial oxoglutarate carrier that was otherwise devoid of cysteine residues. The effects of the cysteine replacement and subsequent modification by sulfhydryl reagents on the initial uptake rate of 2-oxoglutarate were determined. The results were evaluated using a structural model of the oxoglutarate carrier. Residues involved in inter-helical and lipid bilayer interactions tolerate cysteine replacements or their modifications with little effect on transport activity. In contrast, the majority of cysteine substitutions in the aqueous cavity had a severe effect on transport activity. Residues important for function of the carrier cluster in three regions of the transporter. The first consists of residues in the [YWLF]- [KR]-G-X-X-P sequence motif, which is highly conserved in all members of the mitochondrial carrier family. The residues may fulfill a structural role as a helix breaker or a dynamic role as a hinge region for conformational changes during translocation. The second cluster of important residues can be found at the carboxy-terminal end of the even-numbered transmembrane alpha-helices at the cytoplasmic side of the carrier. Residues in H6 at the interface with H1 are the most sensitive to mutation and modification, and may be essential for folding of the carrier during biogenesis. The third cluster is at the midpoint of the membrane and consists of residues that are proposed to be involved in substrate binding.

摘要

线粒体酮戊二酸载体将胞质中的苹果酸与线粒体基质中的2-酮戊二酸进行交换。该载体的直系同源物具有高度的氨基酸序列保守性,这意味着仅基于这一标准无法确定对功能重要的残基。因此,在一个原本不含半胱氨酸残基的功能性线粒体酮戊二酸载体中,将跨膜α-螺旋H2和H6中的每个氨基酸残基替换为半胱氨酸。测定了半胱氨酸替换以及随后巯基试剂修饰对2-酮戊二酸初始摄取速率的影响。使用酮戊二酸载体的结构模型对结果进行了评估。参与螺旋间和脂质双层相互作用的残基能够耐受半胱氨酸替换或其修饰,对转运活性影响很小。相比之下,水腔中的大多数半胱氨酸取代对转运活性有严重影响。对载体功能重要的残基聚集在转运体的三个区域。第一个区域由[YWLF]-[KR]-G-X-X-P序列基序中的残基组成,该基序在线粒体载体家族的所有成员中高度保守。这些残基可能起到螺旋破坏者的结构作用,或者在转运过程中作为构象变化的铰链区域起到动态作用。第二个重要残基簇位于载体胞质侧偶数跨膜α-螺旋的羧基末端。H6中与H1界面处的残基对突变和修饰最为敏感,可能对生物合成过程中载体的折叠至关重要。第三个簇位于膜的中点,由推测参与底物结合的残基组成。

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