Ionic Interactions in Crystalline Bovine Pancreatic Ribonuclease A^,

Abstract

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.

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 A resolution

“…A hydrogen bond between His119 and Asp121 in the wild-type enzyme (de Mel et al 1992) is not essential in favoring the A position because His119 in the F120W mutant does not contain this hydrogen bond but still assumes the A position. An increase in salt concentration or a decrease in pH favors the B position of His119 in the wild-type RNase A (de Mel et al 1994b;Fedorov et al 1996), indicating that a possible ionic interaction between the -electron of Phe120 and the imidazole ring of His119 makes the A position of His119 preferable. Such ionic interaction could be present between the electrons of the indole ring of Trp120 in F120W but not in the F120A and F120G enzymes, which favor the B position over the A position in the crystal.…”

New monoclinic form of bovine pancreatic ribonuclease A from a salt solution and comparison of intermolecular interactions in ribonucleases A

“…Crystallization was carried out by the sitting-drop vapor diffusion or the batch method at 293 K using 0.85-1.5 M ammonium sulfate and 1.5-2.5 M sodium chloride as precipitants in reference to the reported condition of the T form [10]. The molar ratio of sodium chloride to ammonium sulfate was approximately 1.8.…”

“…We used the crystal structure of the T form (PDB [18] code 1RNX [10]) as the search model. In the crystallographic structure refinement using REFMAC5 [19], we applied a non-crystallographic symmetry restraint for four molecules in the asymmetric unit.…”

“…To date, native ribonuclease A (RNase A) is known to crystallize into monoclinic, orthorhombic, hexagonal, and trigonal forms depending on the kinds of precipitants such as salts, alcohols, and PEG [7][8][9][10][11][12][13]. The new crystal form was found in the course of examining the crystal growth of trigonal RNase A (hereafter, the known trigonal form is called T form) using ammonium sulfate and sodium chloride as precipitants.…”

Crystallographic and functional studies of a modified form of eosinophil-derived neurotoxin (EDN) with novel biological activities

“…Conformation A is compatible with nucleotide binding, 19 ± 21 whereas low pH or the presence of sulfate/phosphate in the active site favors conformation B. 14,22 The high-resolution structure of (À4)EDN contains both of these conformations of His129. The occupancy of conformation B is 0.61 with an average B-factor of 16.2 A Ê 2 , whereas the occupancy of conformation A is 0.39 with an average B-factor of 16.6 A Ê 2 .…”