Productive and nonproductive binding to ribonuclease A: X‐ray structure of two complexes with uridylyl(2′,5′)guanosine

Abstract

Guanine-containing mono-and dinucleotides bind to the active site of ribonuclease A in a nonproductive modẽ retro-binding!~Aguilar CF, Thomas PJ, Mills A, Moss DS, Palmer RA, 1992, J Mol Biol 224:265-267!. Guanine binds to the highly specific pyrimidine site by forming hydrogen bonds with Thr45 and with the sulfate anion located in the P1 site. To investigate the influence of the anion present in the P1 site on retro-binding, we determined the structure of two new complexes of RNase A with uridylyl~29,59!guanosine obtained by soaking two different forms of pre-grown RNase A crystals. In one case, RNase A was crystallized without removing the sulfate anion strongly bound to the active site; in the other, the protein was first equilibrated with a basic solution to displace the anion from the P1 site. The X-ray structures of the complexes with and without sulfate in P1 were refined using diffraction data up to 1.8 Å~R-factor 0.192! and 2.0 Å~R-factor 0.178!, respectively. The binding mode of the substrate analogue to the protein differs markedly in the two complexes. When the sulfate is located in P1, we observe retro-binding; whereas when the anion is removed from the active site, the uridine is productively bound at the B1 site. In the productive complex, the electron density is very well defined for the uridine moiety, whereas the downstream guanine is disordered. This finding indicates that the interactions of guanine in the B2 site are rather weak and that this site is essentially adenine preferring. In this crystal form, there are two molecules per asymmetric unit, and due to crystal packing, only the active site of one molecule is accessible to the ligand. Thus, in the same crystal we have a ligand-bound and a ligand-free RNase A molecule. The comparison of these two structures furnishes a detailed and reliable picture of the structural alterations induced by the binding of the substrate. These results provide structural information to support the hypotheses on the role of RNase A active site residues that have recently emerged from site-directed mutagenesis studies.

The Importance of Dynamic Effects on the Enzyme Activity

“…Although small, this movement places the side chain in a more favorable position to activate the enzymatic reaction. Incidentally a similar hydrogen bond was found in RNase A between the conserved active site residues Lys 41 and Asn 44 (41). The features observed in the room temperature structure of M23L-ONC were fully confirmed by the 100 K structure refined at a resolution of 1.5 Å.…”

“…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).…”

“…Another distinct property of ONC is the presence of an N-terminal pyroglutamate (Pyr 1 ), which folds back against the N-terminal helix (13). The overall active site architecture closely resembles that of RNase A and includes His 10 , Lys 31 , and His 97 , which form the catalytic triad corresponding to His 12 , Lys 41 , and His 119 , respectively, of the pancreatic enzyme. Despite this similarity and the activating effect of the N-terminal Pyr residue (14), the Onconase activity is by 3-5 orders of magnitude lower in comparison with RNase A, depending on the used substrate (15).…”

A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish

“…The map of the anionic binding sites is strictly conserved in the structure at neutral pH, with the only exception of the acetate that is replaced by an additional sulphate anion. Thus, in contrast to numerous crystallographic reports on RNase A [15,18] and other members of the family [19][20][21], and angiogenins [16,17], and despite the relatively high concentration in the crystallization mixture, none of the sulphate ions is located in the active site, as the anion binds preferentially to other regions of ZF-RNase-1. The putative binding subsite B1 of the pyrimidine base is partially obstructed by the side chain of Glu122 located in the C-terminal segment of the protein.…”

Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme

“…The relevance of vanadiumoxide binding in the ribonuclease (RNase) A active site [10,12] has been challenged, since amino acids poised for transition-state stabilization did not support roles ascribed by biochemical and kinetic analyses [13]. Complementary studies of RNases have been conducted in the presence of substrate analogs comprising 3′-OH, 2′,5′-linkages at the site of cleavage [14][15][16]. Several such structures were consistent with expectations for transition-state stabilization including placement of positively charge functionalities near the leaving group, and hydrogen-bond donors at the non-bridging oxygens of the scissile bond.…”