Structural Basis for the Biological Activities of Bovine Seminal Ribonuclease

Abstract

Bovine seminal ribonuclease (BS-RNase) is a homolog of RNase A with special biological properties that include specific antitumor, aspermatogenic, and immuno-suppressive activities. Unlike RNase A, BS-RNase is a dimer cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. At equilibrium, this dimer is a mixture of two distinct quaternary forms, M = M and M x M. The conversion of M = M to M x M entails the exchange of NH2-terminal alpha-helices between subunits. Here, the cytotoxic activities of purified M x M were shown to be greater than those of purified M = M, despite extensive equilibration of M = M and M x M during the time course of the assays. Replacing Cys31 or Cys32 with a serine residue did not compromise the enzymatic activity of dimeric BS-RNase, but reduced both the fraction of M x M at equilibrium and the cytotoxicity. We conclude that the M x M form is responsible for the special biological properties of BS-RNase. Since cytosolic ribonuclease inhibitor binds tightly to monomeric but not dimeric BS-RNase and only the M x M form can remain dimeric in the reducing environment of the cytosol, we propose that BS-RNase has evolved its M x M form to retain its lethal enzymatic activity in vivo.

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

“…The electron density associated with the two hinge regions (residues [16][17][18][19][20][21][22] is well defined [ Fig. 1(C)].…”

“…Moreover, the remaining part of the hinge loop is characterized by a conformation of Gly16 not accessible to a non-Gly residue (Asp in HHP2-RNase) and stabilized by a hydrogen bond with the Arg80 side chain. 19 In the seminal enzyme the different properties of the M¼M and MxM forms have been rationalized in terms of their behavior under reducing conditions 16,22 : the former yields a monomeric enzyme, whereas the latter gives rise to a metastable noncovalent dimer (NCD-BSRNase), characterized by a structure similar to that of the parent species. The possibility that a similar noncovalent transient dimeric species could be formed also for HHP2-RNase has been investigated using a docking procedure inspired by the 3D structures of NCD-BSRNase.…”

“…17 They differ for the swapping of the N-termini (residues 1-15) within a substantially identical quaternary framework. [18][19][20] In both isoforms, the dimeric interface is formed by the hinge peptides (residues [16][17][18][19][20][21][22] and the helices (residues [23][24][25][26][27][28][29][30][31][32][33][34] that comprise the four cysteines forming the two interchain disulfide bridges (Cys31-Cys32 0 and Cys32-Cys31 0 ) and the side chains of Leu28 and 28 0 that form stabilizing hydrophobic interactions across the molecular twofold axis. 18,20 In the most abundant swapped form, MxM-BSRNase, the dimeric interface also includes the closed interface arising from the swapped elements.…”

“…16 In addition, the proximity of the cysteine residues may enhance the formation of the disulfide bridges and explain the higher stability of the covalent linkages in the swapped protein with respect to the unswapped one. 16,22 To obtain a potential chemotherapeutic agent more efficiently tolerated by the human immune system, various attempts have been made to transfer the antitumor structural determinants of ONC and BSRNase to human pancreatic ribonuclease (HPRNase), a monomeric enzyme very sensible to RI inactivation. 11,23 HP-RNase, whose physiological role remains unclear, possesses 70% identity with both bovine pancreatic and seminal enzymes.…”

“…35 Both HHP-RNase and HHP2-RNase fold in the unswapped and swapped forms, the latter being considerably less abundant. 35 On the basis of BSRNase data, 16,22 the high antitumor action exhibited by the unswapped form of these mutants is surprising, especially because the complex between RI and the monomeric enzyme HP-RNase was found to be extremely stable (K d ¼ 2.9 Â 10 À16 M and t 1/2 ¼ 81 days). 29 The cytoxic activity of the unswapped dimers has been ascribed to the unusual stability of the interchain disulfide bridges that makes them more similar to MxM-BSRNase and allows their survival as dimers in the cytosolic compartment.…”