New TSPO Crystal Structures of Mutant and Heme-Bound Forms with Altered Flexibility, Ligand Binding, and Porphyrin Degradation Activity.
New TSPO Crystal Structures of Mutant and Heme-Bound Forms with Altered Flexibility, Ligand Binding, and Porphyrin Degradation Activity.
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DOI：
10.1021/acs.biochem.2c00612
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发表时间：
2023-04-04
期刊：
影响因子：
2.9
通讯作者：
Ferguson-Miller, Shelagh
中科院分区：
文献类型：
--
作者：
Liu, Jian;Hiser, Carrie;Li, Fei;Hall, Robert;Garavito, R. Michael;Ferguson-Miller, Shelagh
古老的蛋白质 TSPO（易位蛋白 18kD）在所有王国中都有发现，最初被确定为苯二氮卓类药物的结合位点。尽管卟啉是保守的配体，但其生理功能仍不清楚。细菌 TSPO 的几种晶体结构和小鼠形式的核磁共振结构已经揭示了单体和二聚体构型，但尚未有关于具有生理配体的结构的报道。在这里，我们展示了与生理配体结合的球形红杆菌 TSPO 的第一个 X 射线结构。两种不同的变体（用苏氨酸取代位置 139 处的丙氨酸（A139T）和用苯丙氨酸取代位置 138 处的丙氨酸（A138F））产生了良好衍射的晶体，给出了含脱辅基和含血红素形式的结构。两种变体对血红素和原卟啉 IX 都具有野生型微摩尔亲和力，但 A139T 在光和氧存在下加速卟啉分解的能力非常低。血红素与任一突变体的二聚体的一个原聚体的结合在血红素结合原聚体和未结合血红素的原聚体中诱导更刚性的结构，证明了变构反应。 X 射线数据的整体细化揭示了响应单个血红素与二聚体结合而改变的灵活性的不同区域。 A139T 变体总体上显示出更刚性的结构，这可能与血红素缝隙中捕获的水的额外氢键有关。由于 TSPO 被认为在从线粒体到细胞质的血红素递送中发挥作用，因此新结构提供了有关此类活性的结构基础的潜在线索。
The ancient protein TSPO (translocator protein 18kD) is found in all kingdoms and was originally identified as a binding site of benzodiazepine drugs. Its physiological function remains unclear, although porphyrins are conserved ligands. Several crystal structures of bacterial TSPO and nuclear magnetic resonance structures of a mouse form have revealed monomer and dimer configurations, but there have been no reports of structures with a physiological ligand. Here, we present the first X-ray structures of Rhodobacter sphaeroides TSPO with a physiological ligand bound. Two different variants (substituting threonine for alanine at position 139 (A139T) and phenylalanine for alanine at position 138 (A138F)) yielded well-diffracting crystals giving structures of both apo- and heme-containing forms. Both variants have wild-type micromolar affinity for heme and protoporphyrin IX, but A139T has very low ability to accelerate the breakdown of porphyrin in the presence of light and oxygen. The binding of heme to one protomer of the dimer of either mutant induces a more rigid structure, both in the heme-binding protomer and the protomer without heme bound, demonstrating an allosteric response. Ensemble refinement of the X-ray data reveals distinct regions of altered flexibility in response to single heme binding to the dimer. The A139T variant shows a more rigid structure overall, which may relate to extra hydrogen bonding of waters captured in the heme crevice. As TSPO has been suggested to have a role in heme delivery from mitochondria to the cytoplasm, the new structures provide potential clues regarding the structural basis of such activity.
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影响因子：
16.6
作者：
Jaipuria G;Leonov A;Giller K;Vasa SK;Jaremko Ł;Jaremko M;Linser R;Becker S;Zweckstetter M
通讯作者：
Zweckstetter M
影响因子：
7.2
作者：
Frank, Wolfgang;Baar, Kim-Miriam;Reski, Ralf
通讯作者：
Reski, Ralf
影响因子：
2.9
作者：
Guillaumot, Damien;Guillon, Stephanie;Batoko, Henri
通讯作者：
Batoko, Henri
影响因子：
3
作者：
Hiser C;Montgomery BL;Ferguson-Miller S
通讯作者：
Ferguson-Miller S
影响因子：
5.3
作者：
Balsemão-Pires E;Jaillais Y;Olson BJ;Andrade LR;Umen JG;Chory J;Sachetto-Martins G
通讯作者：
Sachetto-Martins G