Wayment-Steele Hannah K, Otten Renee, Pitsawong Warintra, Ojoawo Adedolapo M, Glaser Andrew, Calderone Logan A, Kern Dorothee
Department of Biochemistry, Brandeis University and Howard Hughes Medical Institute, Waltham, MA, USA.
Present address: Treeline Biosciences, Watertown, MA, USA.
bioRxiv. 2024 Jun 19:2024.06.03.597139. doi: 10.1101/2024.06.03.597139.
How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from . Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB's landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.
一个单一的蛋白质结构域如何编码具有多种稳定折叠状态的构象景观，以及这些状态如何相互转化？在这里，我们使用实时和弛豫色散核磁共振来表征来自[具体来源未给出]的昼夜节律蛋白KaiB的构象景观。在已知的天然变构蛋白中独一无二的是，这种KaiB变体在两种单体状态之间自发相互转化：“基态”和“折叠开关”（FS）状态。处于FS状态的KaiB与多个结合伙伴相互作用，包括核心KaiC蛋白，以调节昼夜节律。我们发现KaiB本身在基态和FS状态之间相互转化需要数小时，这突出了单个序列编码功能所需缓慢过程的能力。我们通过证明脯氨酰异构酶CypA加速相互转化，揭示了基态和FS状态之间的限速步骤是折叠开关区域中三个脯氨酸的异构化。相互转化通过一个“部分无序”（PD）状态进行，其中C端一半变得无序，而N端一半保持稳定折叠。我们发现了KaiB景观的另外两个特性。首先，基态经历冷变性：在4°C时，PD状态成为主要存在的状态。其次，基态在毫秒时间尺度上与第四种状态“谜”状态交换。我们结合基于AlphaFold2的预测和核磁共振化学位移预测，预测这种“谜”状态是一种β链寄存器移位，可缓解埋藏的带电残基，并通过实验支持这种结构。这些结果为进化如何设计一个实现其功能所需特定时间的单一序列提供了机制上的见解。
