Trammell M A, Falke J J
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Biochemistry. 1999 Jan 5;38(1):329-36. doi: 10.1021/bi981964u.
Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6'. To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU elements may be found in other receptors and signaling proteins.
配体与大肠杆菌和鼠伤寒沙门氏菌的同二聚天冬氨酸受体结合会产生跨膜信号，该信号调节细胞质组氨酸激酶的活性，从而控制细胞趋化性。该受体还能感知细胞内pH值和环境温度，并通过一种适应系统进行共价修饰。受体细胞质结构域中的一个特定螺旋，即α6螺旋，先前已被认为与这些多种输入信号的处理有关。虽然α6螺旋暴露于溶剂的表面具有已知在激酶调节中起作用的适应性甲基化位点，但其埋藏面的功能意义尚不清楚。这个埋藏区域位于亚基界面，α6螺旋与其对称伙伴α6'螺旋相互堆积。为了测试α6螺旋-α6'螺旋界面在激酶调节中的作用，本研究在α6螺旋埋藏面的甘氨酸278位置引入了一系列13个侧链取代。观察到这些取代在体内和体外显著改变受体功能，产生的效应范围从激酶超激活（11个例子）到完全激酶抑制（1个例子）。此外，四个疏水的支链侧链（缬氨酸、异亮氨酸、苯丙氨酸和色氨酸）将激酶锁定在超激活状态，无论受体是否被配体占据。在位置278处的大多数侧链取代产生激酶超激活的观察结果，加上在其他受体位置这种容易的超激活很少见的证据，确定埋藏的甘氨酸278残基为一个调节热点，在这里螺旋堆积与激酶调节紧密耦合。总之，α6螺旋及其堆积相互作用起到一个简单的中央处理单元（CPU）的作用，它感知多种输入信号，整合这些信号，并将输出传递到与组氨酸激酶结合的信号亚结构域。类似的CPU元件可能存在于其他受体和信号蛋白中。
