Nature、Cell子刊文章共同解析细胞膜内蛋白酶

Johns Hopkins大学的科学家首次为细胞膜内的酶绘制了“稳定性图谱”，揭开了这种酶的重要形态维持区和功能区域。该研究陆续发表在Cell子刊Structure杂志网络版和Nature Chemical Biology杂志上。

　　

生物通报道：Johns Hopkins大学的科学家首次为细胞膜内的酶绘制了“稳定性图谱”，揭开了这种酶的重要形态维持区和功能区域。该研究陆续发表在Structure杂志网络版和Nature Chemical Biology杂志上，研究人员希望通过他们的研究推动疟疾及其他寄生虫病的治疗。

“这是首次真正了解这种酶结构背后的建筑逻辑，”Johns Hopkins大学医学院分子生物学和遗传学副教授Sinisa Urban博士说，他的研究团队对一类位于细胞膜内的特殊酶，菱形蛋白酶rhomboid proteases，进行了研究。

膜内蛋白酶在细胞膜中水解肽键，这一过程在所有生命类型中都普遍存在，并且涉及多种疾病。破解细胞膜内蛋白水解背后的结构支撑非常必要。在多种生物体内存在的菱形蛋白酶位于细胞膜内，并在那里发挥其切割蛋白的作用。

Urban及其同事之前的研究显示，菱形蛋白酶对于引发疟疾的恶性疟原虫非常关键，能帮助它们成功侵入红细胞最终导致感染。研究人员认为，了解菱形蛋白酶形态的稳定性将有助于开发这种酶的抑制物作为治疗药物。“目前菱形蛋白酶还没有相应的选择性抑制剂，”Urban说。“我们非常需要了解该酶的作用机理，它是如石头一般坚硬还是像果冻一样富有弹性？”

菱形蛋白酶研究的挑战在于，这种酶被细胞膜包围使研究人员很难操作。为此，Urban的研究团队采用了一种被称为thermal light scattering的光散射技术，这一技术能在检测分子弹回的光的同时，使酶样品逐渐升温。酶的正常结构一旦受到破坏其散射光就会不同，而此时的温度（即酶的breaking point）反映了酶的内在稳定性。

研究人员对大肠杆菌菱形蛋白酶的稳定性进行了精确地检测。出人意料的是，这种酶比其他与之形态相似的膜蛋白“更像果冻”。研究人员认为这种特性能帮助菱形蛋白酶与其所切割的蛋白相互作用。研究人员合成了150种不同版本的菱形蛋白酶，并分别进行了研究，以了解菱形蛋白酶中哪些是形态维持的重要区域，而哪些区域对于其水解功能至关重要。他们发现了四个维持形态的主要区域和两个重要的功能区。

随后，研究人员应用电脑模拟来对菱形蛋白酶进行分析。他们在菱形蛋白酶的计算机模型中，编入了天然膜环境的特性，这些特性包括主要由脂类组成且水含量极为有限。随后计算机程序模拟了该环境对菱形蛋白酶产生的影响。研究人员发现，菱形蛋白酶中具有一个特殊的内部区域可以存放水分子，这对于这种水解蛋白的酶来说，是在水含量有限的环境中的一大优势。

“这令我们非常兴奋，此外有些稳定性或形态上没有明显改变的菱形蛋白酶却失去了功能，我们对此也非常好奇，”Urban说。他希望更好的了解菱形蛋白酶能有助于引导疟疾等寄生虫病的新药开发。

生物通编辑：叶予

生物通推荐原文摘要：

Architectural and thermodynamic principles underlying intramembrane protease function，Nature Chemical Biology

Intramembrane proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting intramembrane proteolysis is an essential but unattained goal. We integrated new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium unfolding and refolding and quantitative protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid proteases maintain low intrinsic thermodynamic stability (ΔG = 2.1–4.5 kcal mol−1) resulting from a multitude of generally weak transmembrane packing interactions, making them highly responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive 'heat map' of the physiochemical anatomy underlying membrane-immersed enzyme function at, what is to our knowledge, unprecedented resolution.

An Internal Water-Retention Site in the Rhomboid Intramembrane Protease GlpG Ensures Catalytic Efficiency，Structure

Rhomboid proteases regulate key cellular pathways, but their biochemical mechanism including how water is made available to the membrane-immersed active site remains ambiguous. We performed four prolonged molecular dynamics simulations initiated from both gate-open and gate-closed states of Escherichia coli rhomboid GlpG in a phospholipid bilayer. GlpG was notably stable in both gating states, experiencing similar tilt and local membrane thinning, with no observable gating transitions, highlighting that gating is rate-limiting. Analysis of dynamics revealed rapid loss of crystallographic waters from the active site, but retention of a water cluster within a site formed by His141, Ser181, Ser185, and/or Gln189. Experimental interrogation of 14 engineered mutants revealed an essential role for at least Gln189 and Ser185 in catalysis with no effect on structural stability. Our studies indicate that spontaneous water supply to the intramembrane active site of rhomboid proteases is rare, but its availability for catalysis is ensured by an unanticipated active site element, the water-retention site.