Cinar Süleyman, Al-Ayoubi Samy, Sternemann Christian, Peters Judith, Winter Roland, Czeslik Claus
Department of Chemistry and Chemical Biology, TU Dortmund University, D-44221 Dortmund, Germany.
Phys Chem Chem Phys. 2018 Jan 31;20(5):3514-3522. doi: 10.1039/c7cp07399b.
Calmodulin (CaM) is a Ca sensor and mediates Ca signaling through binding of numerous target ligands. The binding of ligands by Ca-saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.
钙调蛋白（CaM）是一种钙传感器，通过与众多靶配体结合来介导钙信号传导。钙饱和的CaM（全CaM）与配体的结合受有吸引力的疏水和静电相互作用支配，这些相互作用在水溶液高压下会减弱。此外，配体结合时潜在的空穴体积形成造成了压力敏感性的另一个来源。因此，高压是探究蛋白质-配体相互作用的合适热力学变量。在本研究中，我们使用X射线和中子散射技术，比较了两种不同配体与全CaM的结合随压力的变化情况。这两种配体分别是K-Ras4B蛋白的法尼基化高变区（HVR），它是全CaM的天然结合伴侣，以及拮抗剂三氟拉嗪（TFP），已知其能抑制全CaM的活性。通过在高达3000巴压力下进行的小角X射线散射实验，我们观察到在没有配体的情况下，游离全CaM会发生压力诱导的部分解折叠，哑铃状蛋白质的两个叶会轻微膨胀。相比之下，与TFP结合时，全CaM形成封闭的球状构象，至少在高达3000巴的压力下是稳定的。K-Ras4B的HVR表现出不同的结合行为，数据表明全CaM/HVR复合物在高压下会解离，这可能是由于与TFP相比，HVR的蛋白质接触密度较低。弹性非相干中子散射实验证实了这些发现。在2000巴以下，压力会导致全CaM/配体复合物中原子波动增强，但全CaM/HVR复合物的波动似乎更大。因此，TFP对全CaM的抑制作用得到了低体积配体结合的支持，尽管这与复合物结构在亚纳秒埃尺度上的刚性化无关。
