A new crystal form of bovine pancreatic RNase A in complex with 2'-deoxyguanosine-5'-monophosphate

Abstract

The structure of bovine pancreatic RNase A has been determined in complex with 2'-deoxyguanosine-5'-monophosphate (dGMP) at 1.33 A resolution at room temperature in a previously unreported unit cell belonging to space group P3(1). There are two molecules of nucleotide per enzyme molecule, one of which lies in the active-site cleft in the productive binding mode, whilst the guanine base of the other dGMP occupies the pyrimidine-specific binding site in a nonproductive mode such that it forms hydrogen bonds to the phosphate group of the first dGMP. This is the first RNase A structure containing a guanine base in the B2 binding site. Each dGMP molecule is involved in intermolecular interactions with adjacent RNase A molecules in the lattice and the two nucleotides appear to direct the formation of the crystal lattice. Because GMP may be produced during degradation of RNA, this association could represent an inhibitor complex and thereby affect the observed enzyme kinetics.

Figure 1

dGMP200 and dGMP300 are shown as bound in the active-site cleft of a molecule of bovine pancreatic RNase A. Nucleotide-binding subsites are labeled B1 (pyrimidine-specific), P1 (hydrolytic site) and B2 (nucleotide base binding site). The electron density from a 2F _o − F _c map from which the dGMP molecules have been omitted, contoured at 1.5σ, is superimposed.

Figure 2

(a) Detailed hydrogen-bonding interactions of dGMP200 with two molecules of RNase A, as well as with dGMP300. (b) The corresponding interactions of dGMP300. The ring-to-ring separation is approximately 3.5 Å between the guanine ring and Tyr115.

Figure 3

Two nucleotides, dGMP200 in yellow and dGMP300 in red, link three RNase A molecules together in the crystal lattice, shown in cartoon representation, from left to right, in green, gray and yellow. Some amino-acid residues important to binding of the nucleotides are emphasized. There are 14 hydrogen-bonding interactions between the nucleotides and the three protein molecules.

Figure 4

Relative positions of the purine rings in (a) structure 1rcn, (b) the present structure and (c) structure 1z6d after superposition of the C^α atoms of the active-site residues His12, Thr45, His119 and Phe120. The active-site residues in the background are displayed as line drawings to establish a point of reference for the positioning of the purine rings in each structure. The interactions for each purine ring are indicated by dashed lines. Residue Asn71 is fairly constantly disposed with respect to the active-site residues, while Gln69 and Glu111 move to accommodate the various base rings of the bound nucleotides at the B2 site. The displacement is approximately in the plane of the adenine ring with a magnitude of ∼2 Å for the guanine ring and of ∼4 Å for the hypoxanthine ring. This demonstrates how the enzyme can accommodate a variety of bases in the B2 site.