Fedorov A A, Joseph-McCarthy D, Fedorov E, Sirakova D, Graf I, Almo S C
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1996 Dec 17;35(50):15962-79. doi: 10.1021/bi961533g.
Isomorphous crystals (space group P3(2)21) of bovine pancreatic ribonuclease A (RNase A) were prepared at a pH of 5.5 in a series of high salt conditions, where both the nature of the ions and the ionic strength varied: 80% ammonium sulfate (mu = 12.5); 8 M sodium formate (mu = 8.0); 3 M NaCl, 30% ammonium sulfate (mu = 7.0); 3 M CsCl, 30% ammonium sulfate (mu = 7.0); and 2.5 M NaCl, 3.3 M sodium formate (mu = 5.8). These structures were independently refined to a resolution of 2.0 A or better with R-factors that range from 16.1% to 17.5%. A comparison of these six structures and the monoclinic crystal form of RNase A grown from alcohol shows that changes in ionic strength do not alter the secondary or tertiary structure and that there are no significant changes in intramolecular salt bridges. These findings support the notion that structures determined from crystals grown in high salt are representative of the overall structural and electrostatic features present under physiological conditions. While little effect was observed on the main chain conformation, several residues adopted different side chain conformations and altered hydrogen-bonding patterns, either as result of direct anion binding or more subtle indirect effects. Changes in the ionic composition of the mother liquor allowed for the occupancy of the active site with different anions. The direct observation of active site-bound chloride and formate anions supports the proposal that these species act as true competitive inhibitors of RNase A and not through nonspecific electrostatic effects. The identification of bound formate anions allowed for an experimental validation of computational-based functional group mapping techniques and suggests a useful modification to these approaches. Electrostatic surface potential calculations identify a nearly continuous band of positive potential, consistent with an extended binding site for polynucleotide ligands and substrates. The majority of these residues are not involved in salt bridges, which may facilitate binding to extended polynucleotide substrates. Selection of the appropriate solvent conditions results in an unoccupied active site, which will allow this crystal form to be used for the crystallographic study of productive ligand-binding modes.
在一系列高盐条件下，于pH 5.5制备了牛胰核糖核酸酶A（RNase A）的同构晶体（空间群P3(2)21），其中离子性质和离子强度均有所不同：80%硫酸铵（μ = 12.5）；8 M甲酸钠（μ = 8.0）；3 M NaCl，30%硫酸铵（μ = 7.0）；3 M CsCl，30%硫酸铵（μ = 7.0）；以及2.5 M NaCl，3.3 M甲酸钠（μ = 5.8）。这些结构被独立精修至2.0 Å或更高分辨率，R因子范围为16.1%至17.5%。对这六种结构与从酒精中生长的RNase A单斜晶体形式的比较表明，离子强度的变化不会改变二级或三级结构，并且分子内盐桥没有显著变化。这些发现支持了这样一种观点，即从高盐中生长的晶体确定的结构代表了生理条件下存在的整体结构和静电特征。虽然在主链构象上观察到的影响很小，但由于直接阴离子结合或更微妙的间接效应，几个残基采用了不同的侧链构象并改变了氢键模式。母液离子组成的变化使得活性位点能够被不同阴离子占据。对活性位点结合的氯离子和甲酸根阴离子的直接观察支持了这样的提议，即这些物质作为RNase A的真正竞争性抑制剂起作用，而不是通过非特异性静电效应。结合的甲酸根阴离子的鉴定使得基于计算的官能团映射技术得到实验验证，并表明对这些方法进行有用的修改。静电表面电位计算确定了一条几乎连续的正电位带，这与多核苷酸配体和底物的扩展结合位点一致。这些残基中的大多数不参与盐桥，这可能有助于与扩展的多核苷酸底物结合。选择合适的溶剂条件会导致活性位点未被占据，这将使这种晶体形式可用于生产性配体结合模式的晶体学研究。
