Lee James, Tomchick Diana R, Brautigam Chad A, Machius Mischa, Kort Remco, Hellingwerf Klaas J, Gardner Kevin H
Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-8816, USA.
Biochemistry. 2008 Apr 1;47(13):4051-64. doi: 10.1021/bi7021156. Epub 2008 Mar 7.
The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatory role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using beta-sheet/beta-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.
枯草芽孢杆菌的KinA蛋白是一种组氨酸蛋白激酶，它能控制该生物体在营养缺乏和其他几种条件下形成芽孢的过程。先前的研究表明，N端的Per-ARNT-Sim结构域（PAS-A）在这种酶的催化活性中起关键作用，这一点通过缺乏该结构域的KinA蛋白自磷酸化速率的显著降低得到了证明。基于PAS结构域在包括其他细菌传感激酶在内的多种蛋白质中所起的环境传感作用，有人提出PAS-A结构域在KinA功能中起重要的调节作用。我们通过结合生物物理和生化方法来研究PAS-A的结构和功能，包括单独研究以及在完整蛋白中的研究，从而探究了这一可能性。在此，我们展示了KinA PAS-A结构域的X射线晶体结构，结果表明它以同二聚体形式结晶，其堆积相互作用为β折叠/β折叠，这与其他几种PAS结构域复合物的情况相同。值得注意的是，我们在晶格中观察到两种具有三级和四级结构差异的二聚体，这表明这些结构域具有显著的结构灵活性。为了证实KinA PAS-A在溶液中也形成二聚体，我们使用了核磁共振光谱、凝胶过滤色谱和分析超速离心相结合的方法，其结果均与晶体学结果一致。我们利用定点诱变实验测试了二聚体界面上几个残基的重要性，发现PAS-A结构域的变化显著改变了KinA在体外和体内的酶活性。这些结果支持了PAS结构域在KinA和其他组氨酸激酶中的重要性，并为天然或人工调节激酶活性提供了可能的途径。
