Koellner G, Choe H W, Heinemann U, Grunert H P, Zouni A, Hahn U, Saenger W
Institut für Kristallographie, Freie Universität Berlin, Germany.
J Mol Biol. 1992 Apr 5;224(3):701-13. doi: 10.1016/0022-2836(92)90554-w.
In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)
在基因发生突变的核糖核酸酶T1 His92Ala（RNase T1 His92Ala）中，活性位点His92的咪唑缺失导致酶失去活性。在用于野生型酶的条件下尝试使RNase T1 His92Ala结晶失败，改进后的方案产生了两种晶体形式，一种是用聚乙二醇（PEG）获得的，另一种是用磷酸盐作为沉淀剂获得的。空间群与野生型RNase T1相同，为P2(1)2(1)2(1)，但晶胞尺寸有显著差异，这与晶体中不同的分子堆积有关；对于PEG衍生的晶体，其尺寸为a = 31.04 Å，b = 62.31 Å，c = 43.70 Å；对于磷酸盐衍生的晶体，尺寸为a = 32.76 Å，b = 55.13 Å，c = 43.29 Å，而未复合的野生型RNase T1的尺寸为a = 48.73 Å，b = 46.39 Å，c = 41.10 Å。通过分子置换法解析晶体结构，并通过立体化学约束最小二乘法进行精修，PEG衍生晶体在10至2.2 Å分辨率范围内基于3712个反射的Fo大于或等于σ(Fo)（R = 15.8%），磷酸盐衍生晶体在10至1.8 Å分辨率范围内基于6258个反射的Fo大于或等于σ(Fo)（R = 14.8%）。His92Ala突变消除了野生型RNase T1中His92NεH...O Asn99的氢键，从而在位于球状酶外围的91至101环中诱导结构灵活性和构象变化。该环在野生型蛋白中由两个β-转角稳定，在用PEG获得的晶体中仅保留了其中一个β-转角。在用磷酸盐作为沉淀剂生长的晶体中，两个β-转角均缺失，并且Gly94 - Ala95 - Ser96 - Gly97片段无序到完全看不到。此外，在两项突变研究中鸟嘌呤结合位点的几何形状与“空的”野生型RNase T1不同，但与在与鸟苷衍生物形成的复合物中发现的相似：Glu46侧链羧酸盐与Tyr42 Oη形成氢键；“空的”野生型RNase T1的鸟嘌呤结合位点中存在的水分子被取代；Asn43 - Asn44肽翻转，使得Asn44的φ/ψ角处于αL构象（当鸟嘌呤结合时在野生型酶中观察到）。（摘要截断于400字）
