Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
J Biol Chem. 2009 Dec 25;284(52):36581-36591. doi: 10.1074/jbc.M109.041343. Epub 2009 Sep 9.
The cAMP receptor protein (CRP) from Mycobacterium tuberculosis is a cAMP-responsive global transcriptional regulator, responsible for the regulation of a multitude of diverse proteins. We have determined the crystal structures of the CRP.cAMP and CRP.N(6)-cAMP derivative-bound forms of the enzyme to 2.2- and 2.3 A-resolution, respectively, to investigate cAMP-mediated conformational and structural changes. The allosteric switch from the open, inactive conformation to the closed, active conformation begins with a number of changes in the ligand-binding cavity upon cAMP binding. These subtle structural changes and numerous non-bonding interactions between cAMP, the N-domain residues, and the C-domain helices demonstrate that the N-domain hairpin loop acts as a structural mediator of the allosteric switch. Based on the CRP.N(6)-cAMP crystal structure, binding of N(6)-cAMP with a bulkier methylphenylethyl extension from the N6 atom stabilizes the cAMP-binding domain, N-domain hairpin, and C-terminal domain in a similar manner as that of the CRP.cAMP structure, maintaining structural integrity within the subunits. However, the bulkier N6 extension of N(6)-cAMP (in R conformation) is accommodated only in subunit A with minor changes, whereas in subunit B, the N6 extension is in the S conformation hindering the hinge region of the central helix. As a result, the entire N-domain and the C-domain of subunit B integrated by the cAMP portion of this ligand, together tilt away ( approximately 7 degrees tilt) from central helix C, positioning the helix-turn-helix motif in an unfavorable position for the DNA substrate, asymmetrically. Together, these crystal structures demonstrate the mechanism of action of the cAMP molecule and its role in integrating the active CRP structure.
结核分枝杆菌的 cAMP 受体蛋白（CRP）是一种 cAMP 反应性的全局转录调节因子，负责调节多种不同的蛋白质。我们已经确定了 CRP.cAMP 和 CRP.N(6)-cAMP 衍生物结合形式的酶的晶体结构，分辨率分别为 2.2 和 2.3 A，以研究 cAMP 介导的构象和结构变化。从开放、无活性构象到封闭、活性构象的变构开关始于 cAMP 结合后配体结合腔中的一系列变化。这些细微的结构变化和 cAMP、N 结构域残基和 C 结构域螺旋之间的众多非键相互作用表明，N 结构域发夹环作为变构开关的结构介导物。基于 CRP.N(6)-cAMP 晶体结构，N6 原子上带有较大的苯乙基延伸的 N(6)-cAMP 的结合以类似于 CRP.cAMP 结构的方式稳定 cAMP 结合域、N 结构域发夹和 C 末端结构域，在亚基内保持结构完整性。然而，N(6)-cAMP 的较大 N6 延伸（在 R 构象中）仅在亚基 A 中以较小的变化被容纳，而在亚基 B 中，N6 延伸在 S 构象中阻碍了中央螺旋的铰链区。结果，整个 N 结构域和 C 结构域通过该配体的 cAMP 部分与亚基 B 一起倾斜（约 7 度倾斜）远离中央螺旋 C，将螺旋-转角-螺旋基序定位在不利于 DNA 底物的不利位置，不对称地。这些晶体结构共同证明了 cAMP 分子的作用机制及其在整合活性 CRP 结构中的作用。
