Yuan T, Vogel H J, Sutherland C, Walsh M P
Department of Biological Sciences, University of Calgary, Alberta, Canada.
FEBS Lett. 1998 Jul 17;431(2):210-4. doi: 10.1016/s0014-5793(98)00750-9.
Most interactions of calmodulin (CaM) with its target proteins are Ca2+-dependent, but a few Ca2+-independent CaM-target protein interactions have been identified. One example is the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We describe here the characterization of the Ca2+-independent interaction between CaM and a synthetic peptide corresponding to the CaM-binding domain of murine macrophage iNOS using circular dichroism (CD) spectroscopy. The CD spectrum of free iNOS peptide indicated a beta-sheet conformation. The interaction of iNOS peptide with apo-CaM in the absence of Ca2+ resulted in the peptide acquiring a type II beta-turn structure. This is in contrast to the situation in the presence of Ca2+ in which case the peptide acquired an alpha-helical conformation upon interaction with CaM, i.e. similar to the Ca2+-dependent interactions of CaM with numerous targets such as myosin light chain kinase (MLCK). Consistent with this similar structural change, iNOS peptide inhibited the Ca2+-CaM-dependent activation of smooth muscle MLCK by competing with MLCK for binding to Ca2+-CaM. The Kd of Ca2+-CaM for iNOS peptide was calculated from competition assays to be 0.3 nM. These results indicate that the structure of the CaM-binding domain of iNOS is quite different when bound to apo-CaM than Ca2+-CaM.
钙调蛋白（CaM）与其靶蛋白的大多数相互作用都依赖于Ca2+，但也已鉴定出少数不依赖Ca2+的CaM-靶蛋白相互作用。一个例子是巨噬细胞中表达的一氧化氮合酶（iNOS）的诱导型同工型。我们在此描述了使用圆二色性（CD）光谱法对CaM与对应于小鼠巨噬细胞iNOS的CaM结合结构域的合成肽之间不依赖Ca2+的相互作用进行的表征。游离iNOS肽的CD光谱表明其具有β-折叠构象。在不存在Ca2+的情况下，iNOS肽与脱辅基CaM的相互作用导致该肽获得II型β-转角结构。这与存在Ca2+时的情况形成对比，在存在Ca2+的情况下，该肽在与CaM相互作用时获得α-螺旋构象，即类似于CaM与许多靶标（如肌球蛋白轻链激酶（MLCK））的Ca2+依赖性相互作用。与这种相似的结构变化一致，iNOS肽通过与MLCK竞争结合Ca2+-CaM来抑制平滑肌MLCK的Ca2+-CaM依赖性激活。通过竞争分析计算出Ca2+-CaM对iNOS肽的Kd为0.3 nM。这些结果表明，iNOS的CaM结合结构域与脱辅基CaM结合时的结构与与Ca2+-CaM结合时的结构有很大不同。
