Abstract
Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16-22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.
Highlights
Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions
At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications
This finding is at variance with what is usually observed in the swapping process, where a monomer to dimer (M/D) transition is commonly observed and the swapped dimer often presents a considerable degree of flexibility and, a certain degree of variability of the end-toend distance of the hinge peptide
Summary
In the panorama of the swapping proteins a special case is represented by bovine seminal ribonuclease (BS-RNase), a homodimeric protein in which the two subunits are covalently linked through two disulfide bridges between cysteines 31 and 32 of one subunit with cysteines 32 and 31 of the partner subunit, respectively [4] In this protein the swapping process involves two dimers, in which the two subunits change their tertiary structure within a basically invariant quaternary assembly imposed by the two interchain disulfides: in the dimerdubbed MxM the N-terminal arms (residues 1–15) are exchanged, or swapped, between the two subunits, whereas in the dimer indicated as MϭM no swapping occurs. We report the x-ray structures of the MxM form of the two mutants and discuss the results on the basis of the MxM/MϭM equilibrium data measured in solution for Ser16-Thr17-Ala19-Ala20-BS-RNase and those previously published for Ala19-BS-RNase and for the parent BS-RNase [12]
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