Cai Mengli, Ying Jinfa, Lopez Juan M, Huang Ying, Clore G Marius
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892-0520.
Departmento de Ciencia-Quimica, Centro de Espectroscopia de Resonancia Magnética Nuclear, Pontificia Universidad Católica del Perú, Lima 32, Perú.
Proc Natl Acad Sci U S A. 2025 May 20;122(20):e2506441122. doi: 10.1073/pnas.2506441122. Epub 2025 May 14.
The bacterial transcriptional regulator RfaH comprises structurally and functionally distinct N- (NTD) and C- (CTD) terminal domains. The latter switches from a helical hairpin packed against the NTD to a five-stranded β-roll upon displacement by RNA polymerase binding. Here, we use exchange-based NMR to probe fold-switching intermediates sampled by the isolated CTD. In addition to the predominant (76 to 77%), semistable β-roll conformation (state A), we identify four structurally and kinetically distinct states: A', B, B', and B″. State B is NMR observable with an occupancy of ~23%, exchanges slowly (τ ~ 300 ms) with the major A species, and comprises a largely unfolded ensemble with transient occupancy of helical (α5*) and β-hairpin (β1*/β2*) elements. Backbone chemical shift-based structure predictions using the program CS-ROSETTA suggest that the two transient structural elements within the B state may interact with one another to form a semicompact structure. A' (0.35%) is an off-pathway state that exchanges rapidly (τ ~ 1 ms) with state A and likely entails a minor localized conformational change in the β1/β2 loop. State B' (0.3%) exchanges rapidly (τ ~ 1.2 ms) with state B and exhibits downfield N backbone shifts (relative to B) in the α5* region indicative of reduced helicity. Finally state B″ (0.05%) exchanges rapidly (τ ~ 0.8 to 1 ms) with either B' (linear model) or B (branched model), displays significant differences in absolute N chemical shift from states B and B', and likely represents a further intermediate with increased helicity along the fold-switching pathway.
细菌转录调节因子RfaH由结构和功能不同的N端（NTD）和C端（CTD）结构域组成。后者在被RNA聚合酶结合取代后，从与NTD堆积的螺旋发夹结构转变为五链β-折叠结构。在这里，我们使用基于交换的核磁共振技术来探测分离的CTD采样的折叠转换中间体。除了占主导地位的（约76%至77%）半稳定β-折叠构象（状态A）外，我们还确定了四种结构和动力学不同的状态：A'、B、B'和B″。状态B通过核磁共振可观察到，占有率约为23%，与主要的A状态缓慢交换（τ约为300毫秒），并且包含一个主要为未折叠的集合体，其中螺旋（α5*）和β-发夹（β1*/β2*）元件有短暂占有率。使用CS-ROSETTA程序基于主链化学位移的结构预测表明，B状态内的两个短暂结构元件可能相互作用形成一个半紧凑结构。A'（约0.35%）是一个偏离路径的状态，与状态A快速交换（τ约为1毫秒），并且可能在β1/β2环中发生轻微的局部构象变化。状态B'（约0.3%）与状态B快速交换（τ约为1.2毫秒），并且在α5*区域表现出相对于B的N主链向低场位移，表明螺旋度降低。最后，状态B″（约0.05%）与B'（线性模型）或B（分支模型）快速交换（τ约为0.8至1毫秒），显示出与状态B和B'相比绝对N化学位移有显著差异，并且可能代表沿着折叠转换途径螺旋度增加的另一个中间体。
