Genome Science and Technology Program, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Structure. 2018 Jul 3;26(7):972-986.e6. doi: 10.1016/j.str.2018.04.018. Epub 2018 May 31.
The Mycobacterium tuberculosis ATP-binding cassette transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). The structures of FHA-1 and FHA-2 were determined by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, respectively. Relative to the canonical 11-strand β-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one β-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (∼50 μM versus 500 μM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, when joined within a polypeptide chain, either FHA domain can bind either linker pThr to form intra- and intermolecular complexes. We hypothesize that this enables tunable phosphorylation-dependent multimerization to regulate Rv1747 transporter activity.
结核分枝杆菌的 ABC 转运蛋白 Rv1747 是细胞壁生物合成中间体的潜在输出蛋白。Rv1747 具有一个由两个 pThr 相互作用的 Forkhead 相关（FHA）结构域组成的细胞质调节模块，通过一个构象无序的连接子连接，连接子上有两个磷酸化受体苏氨酸（pThr）。通过 X 射线晶体学和核磁共振（NMR）光谱学分别确定了 FHA-1 和 FHA-2 的结构。与 FHA-1 的典型 11 链β-三明治 FHA 结构域折叠相比，FHA-2 是环状排列的，缺少一个β-链。然而，两者共享一个保守的 pThr 结合裂缝。FHA-2 比 FHA-1 更不稳定，更具动态性，但与模型 pThr 肽的结合亲和力略高（分别为 50 μM 和 500 μM 的平衡解离常数）。基于 NMR 弛豫和化学位移扰动测量，当连接在多肽链内时，任一 FHA 结构域都可以结合任一连体 pThr，形成分子内和分子间复合物。我们假设这使得可调节的磷酸化依赖性多聚化能够调节 Rv1747 转运蛋白的活性。
