Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
Biochim Biophys Acta Proteins Proteom. 2017 Nov;1865(11 Pt A):1395-1405. doi: 10.1016/j.bbapap.2017.08.011. Epub 2017 Aug 24.
Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp to support DNA replication and repair. The structure of PCNA from Leishmania donovani (LdPCNA) has been determined at 2.73Å resolution. Structure consists of six crystallographically independent molecules which form two trimeric rings. The pore diameter of the individual trimeric ring is of the order of 37Å. The two rings are stacked through their front to front faces. In order to gain a stable packing, the rings are rotated by 42° about the pore axis and shifted by 7Å and tilted by 16° along the perpendicular direction to pore axis. This form of stacking reduced the effective diameter of the pore to 32Å. The sequence of LdPCNA consists of a long segment of 41 amino acid residues (186-Gly-Val-Ser-Asp-Arg-Ser-Thr-Lys-Ser-Glu-Val-Lys-Ala-Glu-Val-Lys-Ala-Glu-Ala-Arg-Asp-Asp-Asp-Glu-Glu-Pro-Leu-Ser-Arg-Lys-Tyr-Gly-Lys-Ala-Asp-Ser-Ser-Ala-Asn-Ala-Ile-226) whereas the corresponding segments in other PCNAs contain only eight residues corresponding to 186-Gly-Val-Ser-Asp-Arg------224-Asn-Ala-Ile-226. The enhanced length of this segment in LdPCNA may influence its mode of interaction with DNA and other proteins. The dissociation constants obtained using real time binding studies with surface plasmon resonance (SPR) for two peptides, Lys-Arg-Arg-Gln-Thr-Ser-Met-Thr-Asp-Phe-Tyr-His (P1) from human cyclin-dependent kinase inhibitor-1(CKI-1) and Lys-Thr-Gln-Gly-Arg-Leu-Asp-Ser-Phe-Phe-Thr-Val (P2) from flap endonuclease 1 (Fen-1) as well as with two small molecule inhibitors, (S)-4-(4-(2-amino-3-hydroxypropyl)-2, 6-diiodophenoxy) phenol hydrochloride (ADPH) and N-(3-methylthiophene-2-carboxylicacid)-N'-((3-hydroxy-2-naphthalenyl) methylene) hydrazide (MCMH) are 0.29±0.09μM, 0.37±0.08μM, 0.35±0.09μM and 1.20±0.08μM respectively. The corresponding values obtained using fluorescence spectroscopic methods were 0.22±0.06μM, 0.68±0.07μM, 0.44±0.07μM and 0.75±0.05μM respectively.
增殖细胞核抗原（PCNA）作为滑动夹支持 DNA 复制和修复。来自利什曼原虫（LdPCNA）的 PCNA 结构已在 2.73Å 分辨率下确定。结构由六个晶体学上独立的分子组成，形成两个三聚体环。单个三聚体环的孔径约为 37Å。两个环通过它们的前面对正面堆叠。为了获得稳定的包装，环围绕孔轴旋转 42°，沿垂直于孔轴的方向移动 7Å 并倾斜 16°。这种堆叠形式将孔的有效直径减小到 32Å。LdPCNA 的序列由 41 个氨基酸残基（186-Gly-Val-Ser-Asp-Arg-Ser-Thr-Lys-Ser-Glu-Val-Lys-Ala-Glu-Val-Lys-Ala-Glu-Ala-Arg-Asp-Asp-Asp-Glu-Glu-Pro-Leu-Ser-Arg-Lys-Tyr-Gly-Lys-Ala-Asp-Ser-Ser-Ala-Asn-Ala-Ile-226）组成，而其他 PCNAs 中的相应片段仅包含 8 个残基对应于 186-Gly-Val-Ser-Asp-Arg------224-Asn-Ala-Ile-226。LdPCNA 中此片段的长度增加可能会影响其与 DNA 和其他蛋白质的相互作用方式。使用表面等离子体共振（SPR）实时结合研究获得的两个肽（来自人细胞周期蛋白依赖性激酶抑制剂-1（CKI-1）的 Lys-Arg-Arg-Gln-Thr-Ser-Met-Thr-Asp-Phe-Tyr-His（P1）和来自核酸内切酶 1（Fen-1）的 Lys-Thr-Gln-Gly-Arg-Leu-Asp-Ser-Phe-Phe-Thr-Val（P2））的解离常数，以及两种小分子抑制剂（（S）-4-（4-（2-氨基-3-羟基丙基）-2,6-二碘苯氧基）苯酚盐酸盐（ADPH）和 N-（3-甲基噻吩-2-羧酸）-N'-（（3-羟基-2-萘基）亚甲基）酰肼（MCMH））分别为 0.29±0.09μM、0.37±0.08μM、0.35±0.09μM 和 1.20±0.08μM。使用荧光光谱法获得的相应值分别为 0.22±0.06μM、0.68±0.07μM、0.44±0.07μM 和 0.75±0.05μM。
