Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity.增殖细胞核抗原（Proliferating Cell Nuclear Antigen, PCNA）是DNA复制与修复的必需因子。PCNA由三个相同单体对称结合形成分子量为90 kDa的环状盘状结构。该环可环绕DNA，并作为聚合酶与其他蛋白的停靠平台，以介导不同DNA代谢过程的进行。人类PCNA的氨基酸序列与酵母同源蛋白的同源性为35%，且二者的三维晶体结构完全一致。本研究证实，酿酒酵母（budding yeast, sc）与人类（human, h）PCNA在溶液中的结构高度相似，但稳定性与动态特性存在显著差异。相较于酿酒酵母PCNA（scPCNA），人类PCNA（hPCNA）对化学变性与热变性的耐受性更弱，且解折叠协同性更低。溶剂交换速率测定结果显示，交换速率最慢的骨架酰胺残基位于结构核心的β折叠片层中，而非排布于中央通道的螺旋区域。然而，人类PCNA的所有骨架酰胺均快速交换，数小时内便无法被检测到；而酿酒酵母PCNA核心区域酰胺的信号则可维持更久。在与DNA结合的PCNA构象中，面向DNA的α螺旋具有较高的动态性，这可能对PCNA环在DNA上的滑动具有重要功能意义：带有柔性内壁的大孔洞可促进短暂且易解离的蛋白质-DNA相互作用的形成。相较于scPCNA，hPCNA的动态性更强，这使其在结合底物时可获得多种诱导构象，从而扩大其结合多样性。