Joglekar Isha, Giridhar Mithun Nag Karadi, Diaz David A, Deo Ankit, Clark A Clay
Department of Biology, University of Texas at Arlington, Arlington, Texas 76019, U.S.A.
Department of Information Systems, University of Texas at Arlington, Arlington, Texas 76019, U.S.A.
Biochem J. 2025 Aug 5;482(15):1029-46. doi: 10.1042/BCJ20250001.
Apoptotic caspases exist not as static structures but as dynamic ensembles in solution, finely tuned by post-translational modifications and oligomerization. The fine-tuning of this ensemble by cellular cues allows caspases to influence not only apoptotic pathways but also the non-apoptotic pathways in which they are involved. These ensembles span a complex conformational landscape from well-characterized low-energy states captured in structural databases to transient high-energy intermediates that remain elusive and poorly understood. This limited structural view poses a major barrier to fully understanding how caspase activity is regulated and diversified across cellular contexts. To address this, we integrate evolutionary, folding, and mutational data with molecular dynamics simulations and network analysis to uncover a highly conserved residue network in structural space that has been faithfully passed on in sequence space over 500 million years of vertebrate evolution. This network encodes a high-energy intermediate consistently present in the ensemble of all present-day vertebrate apoptotic caspases. It not only guides folding but also scaffolds dynamic motions, functioning like a structural backbone that supports the ensemble. Building on this foundation, we identify differentially evolving networks surrounding the conserved core in initiator and effector caspase subfamilies. These variations provide thermodynamic insight into how initiators stabilize monomeric conformations while effectors favor dimeric states, revealing how evolution shapes ensembles to diversify function in protein families. Additionally, we discover conserved hub residues near an allosteric hotspot, distinct from the core network, that regulate the dynamics of surrounding evolving networks and act as control centers that modulate the conformational equilibrium within the apoptotic caspase ensemble.
凋亡半胱天冬酶并非以静态结构存在，而是以溶液中的动态聚集体形式存在，通过翻译后修饰和寡聚化进行精细调节。细胞信号对这种聚集体的精细调节使半胱天冬酶不仅能影响凋亡途径，还能影响其参与的非凋亡途径。这些聚集体跨越了一个复杂的构象景观，从结构数据库中捕获的特征明确的低能状态到仍然难以捉摸且了解甚少的瞬态高能中间体。这种有限的结构观点对全面理解半胱天冬酶活性如何在不同细胞环境中受到调节和多样化构成了重大障碍。为了解决这个问题，我们将进化、折叠和突变数据与分子动力学模拟和网络分析相结合，以揭示结构空间中一个高度保守的残基网络，该网络在5亿多年的脊椎动物进化过程中在序列空间中忠实地传递下来。这个网络编码了一种高能中间体，始终存在于所有现代脊椎动物凋亡半胱天冬酶的聚集体中。它不仅指导折叠，还为动态运动搭建支架，其功能就像支撑聚集体的结构骨架。在此基础上，我们确定了起始和效应半胱天冬酶亚家族中围绕保守核心的差异进化网络。这些变异为起始半胱天冬酶如何稳定单体构象而效应半胱天冬酶倾向于二聚体状态提供了热力学见解，揭示了进化如何塑造聚集体以使蛋白质家族中的功能多样化。此外，我们在一个变构热点附近发现了保守的枢纽残基，与核心网络不同，它调节周围进化网络的动力学，并作为控制中心调节凋亡半胱天冬酶聚集体内的构象平衡。
