Sandberg Anders, Johansson Denny G A, Macao Bertil, Härd Torleif
Department of Medical Biochemistry, Göteborg University, PO Box 440, SE-405 30 Göteborg, Sweden.
J Mol Biol. 2008 Apr 4;377(4):1117-29. doi: 10.1016/j.jmb.2008.01.051. Epub 2008 Jan 30.
A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G(-1)S+1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N-->O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t((1/2))) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by approximately 10 kcal mol(-1) compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to approximately 11 and approximately 15 kcal mol(-1), respectively, but approximately 7 kcal mol(-1) of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of approximately 7 kcal mol(-1) was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G(-1)A+1VVV motif) and the wild-type protein. The results imply that cleavage by N-->O acyl shift alone would proceed with a t((1/2)) of approximately 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.
具有SEA（海胆精子蛋白、肠激酶和集聚蛋白）结构域折叠的一类蛋白质以异源二聚体形式存在，该异源二聚体由特征性G(-1)S+1VVV序列内的自蛋白水解切割产生。自蛋白水解通过丝氨酸羟基对邻近甘氨酸羰基的亲核攻击发生，随后进行N→O酰基转移以及所得酯的水解。有人认为，蛋白质折叠时可裂解肽键的张力会加速该反应。在随附的一篇文章中，我们报道了其机制；在本文中，我们提供了进一步的关键证据，并阐述了蛋白质折叠与自蛋白水解偶联的能量学。GPR116结构域和MUC1 SEA结构域的切割半衰期（t((1/2))）值分别为12分钟和18分钟，与未催化的水解相比，活化能垒的自由能降低了约10 kcal mol(-1)。测得GPR116和MUC1 SEA结构域的解折叠自由能分别约为11 kcal mol(-1)和约15 kcal mol(-1)，但约7 kcal mol(-1)的构象能以前体中可裂解肽键上的张力形式存在，通过底物去稳定化来催化自蛋白水解。通过使用预平衡模型分析核心突变或添加尿素使GPR116 SEA结构域不稳定时获得的稳定性和解切割动力学数据，以及MUC1 SEA前体突变体S1098A（具有G(-1)A+1VVV基序）与野生型蛋白的热力学稳定性差异，测得约7 kcal mol(-1)的张力能。结果表明，仅通过N→O酰基转移进行切割时，t((1/2))约为2.3年，这太慢而无法产生生化效应。随后对其他自切割蛋白的结构数据进行回顾表明，可裂解肽键的构象张力可能是自蛋白水解的常见机制。
