Mirza Mahmooda, Robinson Paul, Kremneva Elena, Copeland O'neal, Nikolaeva Olga, Watkins Hugh, Levitsky Dimitry, Redwood Charles, El-Mezgueldi Mohammed, Marston Steven
National Heart and Lung Institute, Imperial College London, London SW3 6LY, United Kingdom.
J Biol Chem. 2007 May 4;282(18):13487-97. doi: 10.1074/jbc.M701071200. Epub 2007 Mar 13.
E40K and E54K mutations in alpha-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser alpha-tropomyosin (AS-alpha-Tm) expressed in Escherichia coli, that both mutations decrease Ca(2+) sensitivity. E40K also reduces V(max) of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-alpha-Tm, and the two dilated cardiomyopathy-causing mutants. AS-alpha-Tm has a lower cooperative unit size (6.5) than native alpha-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the K(T) value was the same for all actin-Tm species; however, the K(T) value of actin-Tm-troponin at pCa 5 was 50% less for AS-alpha-Tm E40K than for AS-alpha-Tm and AS-alpha-Tm E54K. K(b), the "closed" to "blocked" equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-alpha-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-alpha-Tm E54K, we observed a unique new domain at 55 degrees C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.
α-原肌球蛋白中的E40K和E54K突变会导致遗传性扩张型心肌病。此前我们利用在大肠杆菌中表达的丙氨酸-丝氨酸α-原肌球蛋白（AS-α-Tm）发现，这两种突变都会降低Ca(2+)敏感性。E40K还会使肌动蛋白-Tm激活的S-1 ATP酶的V(max)降低18%。我们研究了天然Tm、AS-α-Tm以及两种导致扩张型心肌病的突变体的协同变构调节。AS-α-Tm的协同单位大小（6.5）低于天然α-原肌球蛋白（10.0）。E40K突变将协同单位大小降低至3.7，而E54K则将其增加至8.0。对于On状态和Off状态之间的平衡，所有肌动蛋白-Tm种类的K(T)值相同；然而，在pCa 5时，AS-α-Tm E40K的肌动蛋白-Tm-肌钙蛋白的K(T)值比AS-α-Tm和AS-α-Tm E54K低50%。K(b)，即“关闭”到“阻断”的平衡常数，对于所有原肌球蛋白种类都相同。E40K突变使原肌球蛋白对肌动蛋白的亲和力降低了1.74倍，但仅在On状态（存在S-1时）。相比之下，E54K突变仅在Off状态下使亲和力降低了3.5倍。对AS-α-Tm的差示扫描量热法测量表明，位于原肌球蛋白N端的结构域3因这两种突变而强烈不稳定。此外，对于AS-α-Tm E54K，我们在55℃观察到一个独特的新结构域，其占焓的25%，表明原肌球蛋白的一部分得到了稳定。E40K突变的致病机制可能是由于细肌丝On状态的不稳定；然而，E54K突变对原肌球蛋白的结构和功能有更复杂的影响。
