The dual-mode quaternary structure of seminal RNase.

Abstract

Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.Bovine seminal ribonuclease (BS-RNase) is a homodimeric ribonuclease-in fact, the only dimeric RNase isolated thus far. Its enzymic properties are unusual for a ribonuclease, as it cleaves effectively single-and double-stranded RNA and is allosterically regulated in the rate-determining step of the reaction. These properties are matched by its unusual biological, noncatalytic actions, including its selective toxicity toward tumor cells, activated T cells, and the male germ cell line (ref. 1 and references cited therein).In the structure of naturally dimeric BS-RNase determined by x-ray crystallography (2, 3), the two subunits interchange their N-terminal segments (see Fig. 1A) as do the monomers of artificially dimerized bovin' pancreatic RNase (RNase A) in the structure proposed by Crestfield and others (4, 5). A significant consequeoce of ths 'structural arrangement is the composite nature 9f the active sites of dimerized RNase A and of naturally dimeric BS-RNase. Composite, shared active sites have been previously found for a few oligomeric enzymes (6-9). However, in all of these cases, dissociation of the oligomers produced inactive monomers. In contrast, isolated BS-RNase monomers are active-in fact, more active than the parent dimeric enzyme, although they lack its typical allosteric properties (10).The dimeric structure of BS-RNase is maintained not only by noncovalent interactions between subunits (3) but also by two intersubunit disulfides, which link Cys-31 and Cys-32 of one subunit with Cys-32 and Cys-31, respectively, of the other subunit (11, 12). We have previously observed (12) that selective reduction of the intersubunit disulfides monomerizes only a fraction of the dimeric protein. This observation, which had not been fully understood thus far, has since been confirmed repeatedly, also in other laboratories (13), with protein preparations obtained with different purification ...

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

“…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.…”

“…Sequencing grade trypsin, modified by reductive methylation to reduce autolysis, was obtained from Promega (Madison, Wisconsin). BS-RNase quaternary forms (MXM and M=M) were isolated following the protocol described previously (Piccoli et al, 1992). Reagents were purchased from Sigma (St. Louis, Missouri).…”

“…Protein concentration was determined by amino acid analysis and a colorimetric assay (BCAProtein Assay, Pierce, Rockford, Illinois). The models of 17K(MXM) and 17K(M=M) were derived with the MOLSCRIPT program (Kraulis, 1991) from the structure of BS-RNase in the MXM form (PDM codename lBSR), and from a model of M=M (Piccoli et al, 1992), respectively.…”

“…been described for the protein (Piccoli et al, 1992). The native enzyme is, in fact, an equilibrium mixture of MXM and M=M dimers, in a molar ratio of 2:l.…”

“…ered that the displacement of the N-terminal a-helix might occur in the interconversion of BS-RNase quaternary forms under equilibrium conditions (Piccoli et al, 1992).…”