title: Novel non-productively bound ribonuclease inhibitor complexes--high resolution X-ray refinement studies on the binding of RNase-A to cytidylyl-2',5'-guanosine (2',5'CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG).
abstract: The X-ray structures of two complexes of bovine ribonuclease-A produced by soaking pre-grown crystals in solutions of the inhibitors cytidylyl-2',5'-guanosine (2',5' CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG) have been determined at 1.5 A resolution and refined by restrained least squares to R = 21.0% for 17,855 reflections, and R = 19.1% for 16,347 reflections, respectively. Binding of the substrate analogs to the protein has taken place in a completely unexpected and previously unreported manner. In each case the guanine base occupies the well characterized B1 pyrimidine binding site adjacent to Thr-45 (described by Richards, F.M., Wyckoff, H.W., Carlson, W.D., Allewell, N.M., Lee, B. and Mitsui, Y. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 35-54, and others including Palmer, R.A., Moss, D.S., Haneef, I. and Borkakoti, N. (1984) Biochim. Biophys. Acta 785, 81-88) having entered through a secondary channel external to the active site itself. We designate this reversed non-productive mode as retro-binding. In this mode of binding the SO4(2-) anion bound in the active site of the native protein crystals (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217) has not been displaced by the phosphate of the inhibitor molecule as originally anticipated and observed in other studies. Instead the CMP or dCMP moiety of the inhibitor molecule is held loosely in a channel running towards the surface of the protein molecule and is thus completely external to the active site. Consequently, although it has been possible to model them, no attempt has been made to refine either the disordered cytosine in the CpG complex or the deoxycytosine in the dCpdG complex. The traditional B2 purine binding site of RNase (Richards et al., 1971) is unoccupied by the soaked inhibitors. Important changes that have taken place in the protein structure include: stabilization of both Lys-41 and Gln-11 via H-bonding to SO4(2-); stabilization of His-119 in the A conformation (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217); and stabilization of SO4(2-) by H-bonds formed with the retro-bound guanine base. Binding of the inhibitors and stabilization of the active site is accompanied by displacement and redistribution of solvent molecules.
authors.affiliation: Department of Crystallography, Birkbeck College, University of London, U.K.
authors.forename: C F
authors.initials: CF
authors.lastname: Aguilar
authors.forename: P J
authors.initials: PJ
authors.lastname: Thomas
authors.forename: D S
authors.initials: DS
authors.lastname: Moss
authors.forename: A
authors.initials: A
authors.lastname: Mills
authors.forename: R A
authors.initials: RA
authors.lastname: Palmer
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003461:Crystallography; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000494:pharmacology*; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014961:X-Ray Diffraction
chemical_list: D015226:Dinucleoside Phosphates; D046911:Macromolecular Substances; D012259:Ribonuclease, Pancreatic