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

Abstract

Dimeric seminal RNase (BS-RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N-terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N-terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N-terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface.Keywords: BS-RNase; domain swapping; limited proteolysis; mass spectrometry; trypsin Bovine seminal RNase (BS-RNase) is an RNase with unusual biological actions, and the only dimeric member of the vertebrate ribonuclease superfamily (D'Alessio et al., 1997). 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.

Trypsin Sheds Light on the Singular Case of Seminal RNase, a Dimer with Two Quaternary Conformations

“…2, B and C). It has been shown previously that a 17-kDa product is the limit digest product for either MxM or MϭM form after a prolonged treatment with trypsin (16).…”

“…Aliquots of 10 g were withdrawn, and the reaction was stopped by adding soybean trypsin inhibitor in a 10-fold excess (w/w) with respect to the enzyme. Products were analyzed by SDS-polyacrylamide (15%) gel electrophoresis (20) or by RP-HPLC (reverse phase high performance liquid chromatography) as described (16).…”

“…Recently, trypsin was tested on the isolated BS-RNase forms and found to be capable of discriminating between MxM and MϭM (16). Different limit digest products (see Fig.…”

“…An aliquot (5 g) of the reduced samples was analyzed by SDS-polyacrylamide (15%) gel electrophoresis (20) to verify that disulfide reduction was complete. Because the monomeric derivative (13.7 kDa) coeluted in the gel filtration pattern with the 17-kDa tryptic product, for an accurate evaluation samples were also analyzed by RP-HPLC, as described (16).…”

Structural Characterization of the M* Partly Folded Intermediate of Wild Type and P138A Aspartate Aminotransferase from Escherichia coli

“…Consequently, when these experiments are performed using a series of proteases with different specificity under conditions that favors a single bond cleavage (complementary proteolysis), the pattern of preferred cleavage sites will depict the exposed regions in the protein molecule (14,15). This strategy was also employed to investigate conformational changes occurring in protein structure under different experimental conditions (16 -19) or during quaternary forms interchange (20,21), as well as for the definition of the interface regions in protein complexes (22)(23)(24).…”