Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C.
Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C.
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酰胺氢揭示了温度依赖性结构转变，可增强心肌肌钙蛋白 C 的 C 结构域突变体中位点 II Ca2 的结合亲和力。
DOI：
10.1038/s41598-017-00777-6
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发表时间：
2017
影响因子：
4.6
通讯作者：
Pinto,JoseR
中科院分区：
文献类型：
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作者：
Veltri,Tiago;deOliveira,GuilhermeAP;Bienkiewicz,EwaA;Palhano,FernandoL;Marques,MayradeA;Moraes,AdolfoH;Silva,JersonL;Sorenson,MarthaM;Pinto,JoseR
肥厚性心肌病相关突变体D145E，位于心肌肌钙蛋白C (cTnC) C结构域，导致分离蛋白多个位点的普遍不稳定。因此，突变体的结构和功能更容易受到高温的影响。在25°C以上，D145E的n结构域Ca2+结合亲和力大幅增加，但野生型蛋白的变化很小。核磁共振衍生的许多残基的主酰胺温度系数在30-40°C以上出现急剧转变，表明在突变的EF-hand IV以及整个C结构域周围，温度依赖的构象变化最为突出。较小的、孤立的变化发生在n域。用D145E重建的心脏皮纤维比用野生型重建的纤维对Ca2+更敏感，这种缺陷在体温附近被放大。我们推测，D145E突变破坏了EF-hand IV的天然构象，导致螺旋H的短暂展开和解离，在较高温度下变得更加突出。这就产生了暴露的疏水表面，可能能够与各种靶标非自然结合，可能包括当cTnC处于开放的Ca2+饱和状态时的n结构域。这将构成从TnC一端向另一端传播信号的潜在途径。
The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.