Atomic resolution structures of ribonuclease A at six pH values

Abstract

The diffraction pattern of protein crystals extending to atomic resolution guarantees a very accurate picture of the molecular structure and enables the study of subtle phenomena related to protein functionality. Six structures of bovine pancreatic ribonuclease at the pH* values 5. 2, 5.9, 6.3, 7.1, 8.0 and 8.8 and at resolution limits in the range 1.05±1.15 A Ê have been re®ned. An overall description of the six structures and several aspects, mainly regarding pH-triggered conformational changes, are described here. Since subtle variations were expected, a thorough validation assessment of the six re®ned models was ®rst carried out. Some stereochemical parameters, such as the NÐC ÐC angle and the pyramidalization at the carbonyl C atoms, indicate that the standard target values and their weights typically used in re®nement may need revision. A detailed comparison of the six structures has provided experimental evidence on the role of Lys41 in catalysis. Furthermore, insights are given into the structural effects related to the pH-dependent binding of a sulfate anion, which mimics the phosphate group of RNA, in the active site. Finally, the results support a number of thermodynamic and kinetic experimental data concerning the role of the disul®de bridge between Cys65 and Cys72 in the folding of RNase A.

The Importance of Dynamic Effects on the Enzyme Activity

“…It is gratifying to notice that both solid state and solution data converge in the indication that the leucine side chain perturbs the lysine native conformation, thus strengthening the indication that this is a genuine feature of the mutant. A similar subtle modification of the side chain conformation of the catalytic Lys 41 was observed in RNase A and was suggested to be important in the substrate binding (41) and in the pH modulation of the enzyme activity (40,46). In the same enzyme a very subtle shift of the active site His 119 was considered to affect the catalytic activity of some mutants of the pancreatic enzyme (47).…”

The Role of the Hinge Loop in Domain Swapping

“…Indeed the torsion angles about the virtual bonds, given in Table III, clearly show the similarity of the peptide conformation with the F conformation of the native enzyme. For comparison, the values of angles for an unswapped conformation of the hinge peptide is also given; since this peptide is disordered in both MϭM (6) and in the monomeric derivative of BS-RNase (29), the conformational parameters presented in Table III are those of RNase A refined at atomic resolution at pH 5.9 (PDB code 1KF3) (31). The hinge peptide of this protein is also shown in Fig.…”

The pH Dependence of Protonation States of Polar Amino Acid Residues Determined by Neutron Diffraction

“…The D 1 atom of His119 is hydrogen bonded to the oxygen of O89 (2.3 Å), while the D 2 atom of His119 is hydrogen-bonded to the O 1 atom of Asp121. It was reported that at pH 6.3, the active site has a sulphate ion, which is hydrogen bonded to the ordered His119, whereas at pH 7.1 the active site releases the sulphate ion and has a disordered His119 (Berisio et al 2002). Although a soaking solution at pD 6.2 was used in this study, the active site does not have the sulphate group, and His119 is ordered.…”

“…To act as a base, His12 should be singly protonated. However, double protonation of His12 at acidic pH has been reported from both X-ray and neutron diffraction analyses (Berisio et al 2002;Wlodawer et al 1983;Wlodawer et al 1986). Neutron diffraction analysis suggested at first glance that His12 is doubly-protonated (positively charged) (Usher et al 1972).…”

“…The structure was initially solved by X-ray diffraction analysis at 5.5 Å resolution (Avey et al 1967). Subsequently, a high resolution (1.05 Å) X-ray diffraction analysis of RNase A was carried out at six different pH values (Berisio et al 2002). Two histidine residues, His12 and His119, play a key role in the cleavage reaction catalyzed by RNase A (Findlay et al 1962;C.…”