Li N, Zhang W, White S W, Kriwacki R W
Department of Structural Biology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 38105, USA.
Biochemistry. 2001 Apr 10;40(14):4293-302. doi: 10.1021/bi0028284.
Bacteriophage T4 encodes a transcription factor, MotA, that binds to the -30 region of middle-mode promoters and activates transcription by host RNA polymerase. The crystal structure of the N-terminal domain of MotA (MotNF) revealed a six-helix domain in which the two C-terminal alpha-helices mediate the formation of a dimer via a coiled-coil motif and hydrophobic interactions. This structure suggested that full-length MotA binds DNA as a dimer, but subsequent biochemical results have shown that a monomeric form of MotA binds DNA. In this study, gel filtration chromatography, dynamic light scattering, and NMR-based diffusion measurements show conclusively that MotNF is a monomer, and not a dimer, in solution. In addition, we have determined the monomeric solution structure of MotNF using NMR spectroscopy, and have compared this with the dimer structure observed in crystals. The core of the protein assumes the same helical conformation in solution and in crystals, but important differences are observed at the extreme C-terminus. In solution, helix alpha5 is followed by five disordered residues that probably link the N-terminal and C-terminal domains of MotA. In crystals, helix alpha5 forms the dimer interface and is followed by a short sixth helix that further stabilizes the dimer configuration. The solution structure of MotNF supports the conclusion that MotA functions as a monomer, and suggests that the existence of the sixth helix in crystals is a consequence of crystal packing. Our work highlights the importance of investigating protein structures in both crystals and solution to fully understand biomolecular structure and to accurately deduce relationships between structure and function.
噬菌体T4编码一种转录因子MotA，它能与中模式启动子的 -30区域结合，并通过宿主RNA聚合酶激活转录。MotA的N端结构域（MotNF）的晶体结构显示为一个六螺旋结构域，其中两个C端α螺旋通过卷曲螺旋基序和疏水相互作用介导二聚体的形成。这种结构表明全长MotA以二聚体形式结合DNA，但随后的生化结果表明MotA的单体形式也能结合DNA。在本研究中，凝胶过滤色谱、动态光散射和基于核磁共振的扩散测量结果确凿地表明，MotNF在溶液中是单体而非二聚体。此外，我们利用核磁共振光谱法确定了MotNF的单体溶液结构，并将其与晶体中观察到的二聚体结构进行了比较。该蛋白的核心在溶液和晶体中呈现相同的螺旋构象，但在极端C端观察到重要差异。在溶液中，α5螺旋后接五个无序残基，这些残基可能连接MotA的N端和C端结构域。在晶体中，α5螺旋形成二聚体界面，其后是一个短的第六螺旋，进一步稳定二聚体构型。MotNF的溶液结构支持MotA以单体形式发挥功能的结论，并表明晶体中第六螺旋的存在是晶体堆积的结果。我们的工作强调了在晶体和溶液中研究蛋白质结构对于全面理解生物分子结构以及准确推断结构与功能关系的重要性。
