Jezewska M J, Bujalowski W
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, 77555-1053, USA.
J Biol Chem. 1996 Feb 23;271(8):4261-5. doi: 10.1074/jbc.271.8.4261.
The direct evidence of dramatic conformational changes of the DnaB hexamer, induced by nucleotide binding, and the presence of multiple conformational states of the enzyme have been obtained by using analytical sedimentation equilibrium, sedimentation velocity studies, and the rigorous fluorescence titration technique. Equilibrium sedimentation measurements show that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its hexameric structure. However, in the presence of the saturating concentration of AMP-PNP, the sedimentation coefficient of the hexamer is s20,w = 11.9 +/- 0.2 compared to the sedimentation coefficient s20,w = 10.5 +/- 0.2 of the free DnaB helicase hexamer. This large sedimentation coefficient change indicates dramatic global conformational transitions of the hexamer, encompassing all six subunits, upon binding the ATP analog. In the presence of ADP, the sedimentation coefficient is s20,w = 11.4 +/- 0.2, indicating that the conformation of the ADP form of the hexamer is different from the ATP form. The sedimentation coefficient of the ternary complex DnaB-(AMP-PNP)-depsilonA(pepsilonA)19, s20,w = 12.4, suggests that the DnaB helicase undergoes further conformational changes upon binding single-stranded DNA (ssDNA). The large global structural changes correlate with the functional activities of the enzyme. In the absence of the ATP analog, the hexamer exists in a "closed" conformation which has extremely low affinity toward ssDNA. Upon binding the ATP analog, the DnaB hexamer transforms into a "tense" state which binds ssDNA with an affinity of approximately 4 orders of magnitude higher than in the absence of the nucleotide. In the presence of ADP, the DnaB hexamer assumes a "relaxed" conformation. The functional difference between these two conformations is reflected in the much weaker allosteric effect of ADP on the ssDNA binding with the affinity constant approximately 3 orders of magnitude weaker than in the presence of the ATP analog (tense state).
通过分析沉降平衡、沉降速度研究以及严格的荧光滴定技术，已获得核苷酸结合诱导DnaB六聚体显著构象变化的直接证据，以及该酶多种构象状态的存在。平衡沉降测量表明，在存在ATP不可水解类似物AMP-PNP的情况下，DnaB解旋酶完全保留其六聚体结构。然而，在存在饱和浓度的AMP-PNP时，六聚体的沉降系数为s20,w = 11.9 +/- 0.2，而游离DnaB解旋酶六聚体的沉降系数为s20,w = 10.5 +/- 0.2。这种较大的沉降系数变化表明，在结合ATP类似物时，六聚体发生了涉及所有六个亚基的显著全局构象转变。在存在ADP的情况下，沉降系数为s20,w = 11.4 +/- 0.2，表明六聚体的ADP形式的构象与ATP形式不同。三元复合物DnaB-(AMP-PNP)-depsilonA(pepsilonA)19的沉降系数s20,w = 12.4，表明DnaB解旋酶在结合单链DNA(ssDNA)时会发生进一步的构象变化。大的全局结构变化与酶的功能活性相关。在不存在ATP类似物的情况下，六聚体以“封闭”构象存在，对ssDNA的亲和力极低。在结合ATP类似物后，DnaB六聚体转变为“紧张”状态，其与ssDNA的结合亲和力比不存在核苷酸时高约4个数量级。在存在ADP的情况下，DnaB六聚体呈现“松弛”构象。这两种构象之间的功能差异反映在ADP对ssDNA结合的变构效应要弱得多，其亲和力常数比存在ATP类似物(紧张状态)时弱约3个数量级。
