Bovine seminal ribonuclease: structure at 1.9 Å resolution

Abstract

The crystal structure of bovine seminal ribonuclease, a homodimeric enzyme closely related to pancreatic ribonuclease, has been refined at a nominal resolution of 1.9/k employing data collected on an electronic area detector. The final model consists of two chains containing 1990 non-H atoms, seven sulfate anions and 113 water molecules per asymmetric unit. The unit-cell parameters are a = 36.5 (1), b = 66.7 (1) and c = 107.5(2)~,, space group P22~2~. The R factor is 0.177 for 16 492 reflections in the resolution range 6.0-1.9 A and the deviations from ideal values of bond lengths and bond angles are 0.020 A and 3.7", respectively. The molecule is formed by two pancreatic like chains, which have their N-terminal segments interchanged so that each active site is formed by residues from both subunits. The two chains are related by a non-crystallographic twofold symmetry and are covalently linked by two consecutive disulfide bridges, which form an unusual sixteen-membered ring across the dimer interface. The deviations from the molecular symmetry, the hydration shell and the sulfate-binding sites are also discussed in relation to the known structure of the pancreatic enzyme.

Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase

“…This loop is partially exposed, and displays a higher temperature factor than the surrounding regions; more importantly, in this loop small differences have been detected between the two chains (Mazzarella et al, 1993). Thus, it can be surmised that the cleavage in one chain is favored for a higher flexibility, hence, a higher affinity of the 35-40 loop in that chain to the trypsin active site.…”

“…In the native MXM form, a second intersubunit interface forms between the exchanged N-terminal a-helix, composed of residues 3-13 and the main body of the partner subunit (Mazzarella et al, 1993). This interface is conserved also in the I7K(MXM) product, as indicated by the finding that in this molecule the two catalytically essential His residues are contributed by distinct chains.…”

“…In Figures 3C and 3D models of the I7K(MXM) and I7K(M=M) products are proposed, based on the three-dimensional structures of MXM (Mazzarella et al, 1993), and of a model of M=M (Piccoli et al, 1992). respectively.…”

“…respectively. Recent results from an X-ray C D In both native dimeric forms of BS-RNase, the interface is formed by the helical segments of the two subunits comprising residues 24-34, and includes the intersubunit disulfides (formed by Cys31 and Cys32) and the hydrophobic interactions (produced by Leu28 and Met29), which hold the two subunits together (Mazzarella et al, 1993). These segments of the original dimeric structures remain undegraded by trypsin and are present in both the 17K(MXM) and the 17K(M=M) tryptic products (see Fig.…”

“…The crystallographic analysis of its structure determined at 1.9 8, resolution (Mazzarella et al, 1993) has shown that: (1) as expected from the high similarity in amino acid sequence (more than SO%), the two identical subunits have a structure very similar to that of RNase A; (2) the two subunits interchange their N-terminal helical segments, giving rise to an intertwined or domain-swapped (Schlunegger et al, 1997) quaternary structure. This structure has been termed the MXM dimeric form of seminal RNase, because a quaternary structure with no interchange of parts (the M=M form) has also Reprint requests to: G.…”