Cytotoxicity of bovine seminal ribonuclease: monomer versus dimer

Bovine seminal ribonuclease (BS-RNase) is a homologue of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, BS-RNase has notable toxicity for human tumor cells. Wild-type BS-RNase is a homodimer linked by two intermolecular disulfide bonds. This quaternary structure endows BS-RNase with resistance to inhibition by the cytosolic ribonuclease inhibitor protein (RI), which binds tightly to RNase A and monomeric BS-RNase. Here, we report on the creation and analysis of monomeric variants of BS-RNase that evade RI but retain full enzymatic activity. The cytotoxic activity of these monomeric variants exceeds that of the wild-type dimer by up to 30-fold, indicating that the dimeric structure of BS-RNase is not required for cytotoxicity. Dimers of these monomeric variants are more cytotoxic than wild-type BS-RNase, suggesting that the cytotoxicity of the wild-type enzyme is limited by RI inhibition following dissociation of the dimer in the reducing environment of the cytosol. Finally, the cytotoxic activity of these dimers is less than that of the constituent monomers, indicating that their quaternary structure is a liability. These data provide new insight into structure-function relationships of BS-RNase. Moreover, BS-RNase monomers described herein are more toxic to human tumor cells than is any known variant or homologue of RNase A including Onconase, an amphibian homologue in phase III clinical trials for the treatment of unresectable malignant mesothelioma.     

Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor

A growing number of pancreatic-type ribonucleases (RNases) present cytotoxic activity against malignant cells. The cytoxicity of these enzymes is related to their resistance to the ribonuclease protein inhibitor (RI). In particular, bovine seminal ribonuclease (BS-RNase) is toxic to tumor cells both in vitro and in vivo. BS-RNase is a covalent dimer with two intersubunit disulfide bridges between Cys(31) of one chain and Cys(32) of the second and vice versa. The native enzyme is an equilibrium mixture of two isomers, MxM and M=M. In the former the two subunits swap their N-terminal helices. The cytotoxic action is a peculiar property of MxM. In the reducing environment of cytosol, M=M dissociates into monomers, which are strongly inhibited by RI, whereas MxM remains as a non-covalent dimer (NCD), which evades RI. We have solved the crystal structure of NCD, carboxyamidomethylated at residues Cys(31) and Cys(32) (NCD-CAM), in a complex with 2'-deoxycitidylyl(3'-5')-2'-deoxyadenosine. The molecule reveals a quaternary structural organization much closer to MxM than to other N-terminal-swapped non-covalent dimeric forms of RNases. Model building of the complexes between these non-covalent dimers and RI reveals that NCD-CAM is the only dimer equipped with a quaternary organization capable of interfering seriously with the binding of the inhibitor. Moreover, a detailed comparative structural analysis of the dimers has highlighted the residues, which are mostly important in driving the quaternary structure toward that found in NCD-CAM.     

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

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2‐RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix‐II (Gln28→Leu, Arg31→Cys, Arg32→Cys, and Asn34→Lys) and to eliminate a negative charge on the surface (Glu111→Gly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well‐defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.     

The buried diversity of bovine seminal ribonuclease: shape and cytotoxicity of the swapped non-covalent form of the enzyme

Bovine seminal ribonuclease exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein. Surprisingly, the cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer. This buried diversity comes into light in the reducing environment of the cytosol, where the unswapped dimer dissociates into monomers, whereas the swapped one generates a metastable dimeric form (NCD-BS) with a quaternary assembly that allows the molecule to escape the protein inhibitor of ribonucleases. The stability of this quaternary shape was mainly attributed to the combined presence of Pro19 and Leu28. We have prepared and fully characterized by X-ray diffraction the double mutant P19A/L28Q (PALQ) of the seminal enzyme. While the swapped and unswapped forms of the mutant have structures very similar to that of the corresponding wild-type forms, the non-covalent form (NCD-PALQ) adopts an opened quaternary structure, different from that of NCD-BS. Moreover, model building clearly indicates that NCD-PALQ can be easily sequestered by the protein inhibitor. In agreement with these results, cytotoxic assays have revealed that PALQ has limited activity, whereas the single mutants P19A and L28Q display cytotoxic activity against malignant cells almost as large as the wild-type enzyme. The significant increase in the antitumor activity, brought about by the substitution of just two residues in going from the double mutant to the wild-type enzyme, suggests a new strategy to improve this important biological property by strengthening the interface that stabilizes the quaternary structure of NCD-BS.     

NMR studies on structure and dynamics of the monomeric derivative of BS-RNase: new insights for 3D domain swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.     

The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases

Bovine seminal ribonuclease (BS-RNase), the only dimeric protein among the pancreatic-like ribonucleases, is endowed with special structural features and with biological functions beyond enzymatic activity. In solution, the protein exists as an equilibrium mixture of two forms, with or without exchange (or swapping) of the N-terminal arms. After selective reduction and alkylation of the two intrachain disulfide bridges, the dimeric protein can be transformed into a monomeric derivative that has a ribonuclease activity higher than that of the parent dimeric protein but is devoid of the special biological functions. A detailed investigation of the structural features of this protein in solution, in comparison with those of other monomeric ribonucleases, may help unveil the structural details which induce swapping of the N-terminal arms of BS-RNase. The solution structure of the recombinant monomeric form of BS-RNase, as determined by 3D heteronuclear NMR, shows close similarity with that of bovine pancreatic ribonuclease (RNase A) in all regions characterized by regular elements of secondary structure. However, significant differences are present in the flexible regions, which could account for the different behavior of the two proteins. To characterize in detail these regions, we have measured H/D exchange rate constants, temperature coefficients and heteronuclear NOEs of backbone amides for both RNase A and monomeric BS-RNase. The results indicate a large difference in the backbone flexibility of the hinge peptide segment 16-22 of the two proteins, which could provide the molecular basis to explain the ability of BS-RNase subunits to swap their N-terminal arms.     

A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.     

A new mutant of bovine seminal ribonuclease with a reversed swapping propensity

Bovine seminal ribonuclease (BS-RNase) is made up of two identical subunits bridged through two disulfide bonds. In solution, it exists as a 2:1 equilibrium mixture between two forms, with (MxM) and without swapping (M=M) of the N-terminal arms. The swapping endows BS-RNase with some special biological functions, including antitumor activity, since MxM retains a dimeric structure even under reducing conditions, thus evading the cytosolic ribonuclease inhibitor. To investigate the structural basis of domain swapping in BS-RNase, we have obtained several mutants by replacing selected residues with the corresponding ones of its monomeric counterpart, bovine pancreatic ribonuclease (RNase A). We have already shown that, in contrast with all other cases of swapped proteins, the swapping propensity of BS-RNase does not depend on the specific sequence of the 16-22 hinge loop, which connects the main body to the dislocating arm. In this paper we report the design, the expression, and the structural characterization of two mutants obtained by replacing Arg80 with Ser either in BS-RNase or in the mutant already containing the 16-22 hinge sequence of RNase A. NMR and circular dichroism data indicate that, in the monomeric form of the latter mutant, Ser80 acts as a switch for the conformation of the hinge region. Accordingly, in the dimeric form of the same mutant the MxM:M=M equilibrium ratio is inverted to 1:2. Overall, these data suggest that the presence of Arg80 triggers the swapping of N-terminal ends and plays a relevant role in the stability of the swapped form of BS-RNase.     

The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease

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.     

The multiple forms of bovine seminal ribonuclease: Structure and stability of a C-terminal swapped dimer

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.     

Engineering the refolding pathway and the quaternary structure of seminal ribonuclease by newly introduced disulfide bridges

Seminal RNase (BS-RNase), a ribonuclease from bovine seminal vesicles, is a homodimeric enzyme with a strong cytotoxic activity selective for tumor cells. It displays the unusual structural feature of existing in solution as an equilibrium mixture of two quaternary isoforms. The major one is characterized by the swap between subunits of their N-terminal ends, whereas the minor isoform shows no swap. The tendency of the two isolated isoforms to interconvert into each other has so far made it difficult to attribute the functional properties of BS-RNase to either isoform. Herein, molecular modeling and site-directed mutagenesis were used to engineer the refolding pathway of BS-RNase and obtain a stable variant of its non-swapping isoform. The protein was engineered with two extra disulfide bridges linking the N-terminal helix of each subunit to the main body of the same subunit. Purified as an active enzyme, the BS-RNase variant was found to be very resistant to thermal denaturation. Its functional characterization revealed that the lack of swapping has a negative effect on the cytotoxic activity of BS-RNase.     

Ribonucleases endowed with specific toxicity for spermatogenic layers

Bovine seminal ribonuclease (BS-RNase) is a dimer in which the subunits are cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. Dimeric BS-RNase is resistant to ribonuclease inhibitor (RI), a protein endogenous to mammalian cells, and is toxic to a variety of cell types. Monomeric BS-RNase (like its homolog, RNase A) is bound tightly by RI and is not cytotoxic. The three-dimensional structure of the RI · RNase A complex suggests that carboxymethylation of C32S BS-RNase (to give MCM31) or C31S BS-RNase (MCM32) could diminish affinity for RI. We find that MCM31 and MCM32 are not only resistant to RI but are also aspermatogenic to mice. In contrast to the aspermatogenic activity of dimeric BS-RNase, that of MCM31 and MCM32 is directed only at spermatogenic layers. Intratesticular injection of MCM31 or MCM32 affects neither the diameter of seminiferous tubules nor the weight of testes. Also, in contrast to wild-type BS-RNase, MCM31 and MCM32 are not toxic to other cell types. Direct immunofluorescence reveals that MCM31 and MCM32 bind only to spermatogonia and primary spermatocytes. This cell specificity makes MCM31 and MCM32 of potential use in seminoma therapy and contraception.     

Double Domain Swapping in Bovine Seminal RNase: Formation of Distinct N- and C-swapped Tetramers and Multimers with Increasing Biological Activities

Bovine seminal (BS) RNase, the unique natively dimeric member of the RNase super-family, represents a special case not only for its additional biological actions but also for the singular features of 3D domain swapping. The native enzyme is indeed a mixture of two isoforms: M = M, a dimer held together by two inter-subunit disulfide bonds, and MxM, 70% of the total, which, besides the two mentioned disulfides, is additionally stabilized by the swapping of its N-termini.When lyophilized from 40% acetic acid, BS-RNase oligomerizes as the super-family proto-type RNase A does. In this paper, we induced BS-RNase self-association and analyzed the multimers by size-exclusion chromatography, cross-linking, electrophoresis, mutagenesis, dynamic light scattering, molecular modelling. Finally, we evaluated their enzymatic and cytotoxic activities.Several BS-RNase domain-swapped oligomers were detected, including two tetramers, one exchanging only the N-termini, the other being either N- or C-swapped. The C-swapping event, confirmed by results on a BS-K113N mutant, has been firstly seen in BS-RNase here, and probably stabilizes also multimers larger than tetramers.Interestingly, all BS-RNase oligomers are more enzymatically active than the native dimer and, above all, they display a cytotoxic activity that definitely increases with the molecular weight of the multimers. This latter feature, to date unknown for BS-RNase, suggests again that the self-association of RNases strongly modulates their biological and potentially therapeutic properties.     

Stability and dynamics of domain-swapped bovine-seminal ribonuclease

The proteins of the ribonuclease-A (RNase-A) family are monomeric, with the exception of bovine-seminal ribonuclease (BS-RNase). BS-RNase is formed by swapping the N-terminal helices across the two monomeric units. A molecular-dynamics (MD) study has been performed on the protein for a simulation time of 5.5 ns to understand the factors responsible for the stability of the dimer. Essential dynamics analysis and motional correlation of the protein atoms yielded the picture of a stabilising, yet flexible, interface. We have investigated the role of intermolecular H-bonding, protein/water interaction, and protein/water networks in stabilising the dimer. The networks of interchain H-bonds involving side-chain/side-chain or side-chain/main-chain (ScHB) interactions between the two chains have also been studied. The ability of protein atoms in retaining particular H2O molecules was investigated as a function of the accessible surface area (ASA), depth, and hydration parameters, as well as their participation in protein/water networks.     

Binding of a substrate analog to a domain swapping protein: X-ray structure of the complex of bovine seminal ribonuclease with uridylyl(2',5')adenosine

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N-terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2',5')adenosine is reported at 2.06 A resolution. The refined model has a crystallographic R-factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, His12, Lys41, His119, and Phe120) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS-RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease

Bovine seminal ribonuclease is a unique case of protein dimorphism, since it exists in two dimeric forms, with different biological and kinetic behavior, which interconvert into one another through three-dimensional swapping. Here we report the crystal structure, at 2.2 A resolution, of the unswapped form of bovine seminal ribonuclease. Besides completing the structural definition of bovine seminal ribonuclease conformational dimorphism, this study provides the structural basis to explain the dependence of the enzyme cooperative effects on its swapping state.     

Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease

Binding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential.     

Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

Thermodynamic stability of the two isoforms of bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase) is a dimeric protein with two identical subunits linked by two disulfide bridges, each subunit showing 80% of sequence identity with pancreatic RNase A. BS-RNase exists in two different quaternary conformations in solution: the MxM form, in which each subunit exchanges its alpha-helical N-terminal segment with its partner, and the M=M form with no exchange. By differential scanning microcalorimetry (DSC), the denaturation of the two dimeric forms of BS-RNase was found to be more complex than a simple two-state process. Monomeric derivatives of the dimeric protein follow instead a simple two-state mechanism, but are distinctly less stable than RNase A. The three-state N if I if D denaturation process of the two quaternary isoforms was interpreted by identifying in the dimers a central highly structured core, enclosing the covalently bonded subunit interface, which unfolds only after the periphery (mainly the N-terminal peptide) unfolds. Circular dichroism spectra of the two forms in the far-ultraviolet region show large differences between the secondary structure of the isoforms and that of the native BS-RNase mixture at equilibrium. This has been attributed to the presence in the equilibrium mixture of intermediate forms with displaced and disordered N-terminal alpha-helical segments.