Dissociation of bovine seminal ribonuclease into catalytically active monomers by selective reduction and alkylation of the intersubunit disulfide bridges.

The hypothesis previously advanced that interchain disulfide bridges link the two identical subunits of bovine seminal ribonuclease BS-1 has been confirmed. The sedimentation rate and the electrophoretic mobility of the protein are not affected by denaturing agents unless thiol reagents are present in the denaturation mixtures. Reduction under controlled conditions results in the immediate cleavage of only 2 disulfide bonds out of 10 percent in the dimeric protein. Under these conditions, and the results do not change when partial reduction is followed by S-alkylation, 30% of the protein dissociates, while the remaining is found to consist of a dimeric species easily dissociable by denaturing agents without addition of thiol reagents. This indicates that the dimeric structure of seminal ribonuclease is maintained not only by disulfide bridges, but also by noncovalent forces. The protein derivative prepared by selective reduction and alkylation has been identified as monomeric bis-S-carboxymethylcysteine-31,32-ribonuclease BS-1. This is on the basis of the characterization of the 14C-labeled S-carboxymethylated peptides isolated from a thermolytic hydrolysate of the derivative prepared with iodo-2-[14C]acetic acid. Monomeric, selectively alkylated ribonuclease BS-1 is stable and catalytically active. The importance of such a derivative is discussed both in the light of the recent studies on the biological actions of seminal ribonuclease and as the fourth component of an experimental system of ribonucleases consisting of two homologous dimers (bovine seminal ribonuclease BS-1 and dimerized bovine pancreatic ribonuclease A) and two homologous monomers (ribonuclease A and the monomeric derivative of ribonuclease BS-1.     

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.     

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

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.     

A calorimetric approach to the study of the interactions of cytidine-3'-phosphate with bovine seminal ribonuclease

A calorimetric study at 25 degrees C is reported on the interaction between allosteric bovine seminal ribonuclease and cytidine-3'-phosphate. The results are compared with those obtained under identical experimental conditions for the interaction of pancreatic ribonuclease A and the same nucleotide. The analysis of the data provides evidence that the binding sites of seminal ribonuclease for cytidine-3'-phosphate are not equivalent, in agreement with previous equilibrium dialysis studies. A model with two sites with different affinities toward the nucleotide, the site with higher affinity resembling the binding site of pancreatic ribonuclease, is proposed. The values calculated for the thermodynamic parameters provide an insight of the forces involved in the interaction of the two enzymes with the nucleotide.     

Intrachain disulfide bridges of bovine seminal ribonuclease

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.     

The unswapped chain of bovine seminal ribonuclease: Crystal structure of the free and liganded monomeric derivative

Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated.     

Study on mammalian ribosomal protein reactivity in situ. III. Effect of trypsin on 40S and 60S subunits.

Rat liver 40S and 60S ribosomal subunits were treated with increasing concentrations of trypsin. The activity of both trypsin-treated subunits, when assayed for polyphenylalanine synthesis, progressively decreased, but the 60S subunits were inactivated at much lower trypsin concentrations than were the 40S ones. The sedimentation coefficients of trypsin-treated subunits were identical to those of control subunits when sucrose gradients containing 0.5 M KCl were used. When the sucrose gradients were prepared with a low salt buffer (80 mM KCl), dimer formation was observed with control subunits, but not with trypsin-treated ones. Two-dimensional gel electrophoresis analysis of the proteins extracted from trypsin-treated subunits revealed that all ribosomal proteins in the subunits were accessible to the enzyme. However, several proteins were more resistant to trypsin in compact subunits than when they were free or in unfolded subunits. Proteins of the 60S subunits were generally digested by lower trypsin concentrations than those of the 40S subunits. From the quantitative measurements of the undigested proteins, a classification of the proteins from both subunits according to their trypsin sensitivity was established. These results were compared with those previously obtained concerning ribosomal protein reactivity to chemical reagents.     

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.     

Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.

Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.     

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.     

Trypanosoma brucei: calcium-dependent endoribonuclease is associated with inhibitor protein

T. brucei cytoplasmic calcium-dependent alkaline ribonuclease activity from DEAE-cellulose fractionation was separated into endoribonuclease and exoribonuclease activities by hydroxyapatite chromatography. T. brucei cytoplasmic extract markedly decreased the endoribonuclease activity, but slightly potentiated the activities of the exoribonuclease and bovine ribonuclease A. While the endoribonuclease was activated by trypsin, the exoribonuclease and bovine ribonuclease A were partially inactivated by trypsin. The endoribonuclease was activated by p-chloromercuribenzoate or N-ethylmaleimide; the exoribonuclease was not affected by these sulfhydryl group reagents. Free ribonuclease was separated from the latent endoribonuclease by 1 M NaCl-Sephadex G-100 gel filtration. The results demonstrate that T. brucei cytoplasm contains a latent endoribonuclease consisting of ribonuclease and inhibitor protein.     

Structural properties of the protein SV-IV.

We have investigated the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of seminal vesicle protein no. 4, a protein with immunomodulatory, anti-inflammatory, and procoagulant activity secreted from the rat seminal vesicle epithelium. The monomeric and trimeric forms were characterized in solution by CD. Details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. The self-association process induces conformational change mainly in the 1-70 region, which appears to be without secondary structure in the monomer but contains alpha-helix in the trimer. In vivo, proteolysis of seminal vesicle protein no. 4 generates active peptides and this is affected by the monomer/trimer state, which is regulated by the concentration of the protein. The information obtained shows how conformational changes between the monomeric and trimeric forms represent a crucial aspect of activity modulation.     

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.     

Helix-destabilization of deoxyribonucleic acid and poly[d(A-T).d(A-T)] by bovine seminal ribonuclease

A ribonuclease isolated earlier from bovine seminal plasma by DNA-affinity chromatography (Ramakrishnamurti, T. and Pandit, M.W. (1983) J. Chromatogr. 260, 216-222) has now been shown by thermal denaturation studies to destabilize the double-helical structure of DNA and poly[d(A-T).d(A-T)]. Thermal denaturation profiles of DNA in the presence of the protein are much more complicated due to the denaturation of protein itself in the temperature range over which DNA predominantly melts. The protein shows relatively stronger affinity towards denatured DNA as compared to native DNA. The action of micrococcal nuclease on DNA and its complexes with ribonuclease A and bovine seminal ribonuclease indicates that both of these proteins destabilize the double-helical structure of native DNA and thereby render the DNA more sensitive to the micrococcal nuclease.     

The effects of cosolutes on protein dynamics: the reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N-oxide, against chemical denaturant was investigated by NMR spin-relaxation measurements and model-free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond-nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N-oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by (1)H-(15)N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.     

An analysis of the reassembly of denatured creatine kinase from monkey brain

Creatine kinase isolated from monkey brain was characterized with respect to denaturation/inactivation and renaturation/reactivation/reassociation in order to determine the mechanism of reassembly. Enzyme unfolded in 8 M urea exhibits several characteristics of denatured protein: complete loss of enzymatic activity, decrease in intrinsic fluorescence with a red shift in the emission maximum and loss of circular dichroism at 220 nm. The renatured protein reassembles to its apparently native condition as judged by these criteria, but small differences of uncertain origin persist. Dependence of activity and fluorescence on denaturant concentration indicate that inactivation is more sensitive to urea than is unfolding; spectral changes at the intermediate urea concentrations suggest formation of aggregated protein. Upon dilution, enzyme previously exposed to 8 M urea for 40 min regains 70-80% native activity, independent of protein concentration over the range of 0.56-160 nM. Reactivation kinetics, measured using the assay mixture with and without trypsin, are independent of protein concentration, and are adequately described by a single rate constant, 3.2 X 10(-3) s-1 and 4.2 X 10(-3) s-1, respectively. Reactivation is completed 20-30 min after initiation of renaturation. Fluorescence changes during refolding are at least biphasic, exhibiting a rapid increase, then a slow decrease completed at approximately 15-20 min after initiating refolding. Reassociation is measured by competitive hybridization between dimerizing B subunits and M subunits to form MB heterodimer. The time dependent decay in heterodimer formation during competitive dimerization shows that reassociation is completed between 60 and 90 min after initiation of reassembly. These results indicate that the brain isozyme of creatine kinase, like the muscle form, is composed of subunits which do not require association for expression of catalytic activity. Furthermore, a comparison of spectral data and susceptibility to trypsin inactivation between the muscle and brain isozymes supports previous suggestions that in the native state, the brain isozyme is a conformationally looser, more open protein.     

Refinement of the structure of bovine seminal ribonuclease

We report here the refinement at 2.5-Å resolution of the x-ray crystal structure of bovine seminal ribonuclease, a dimeric covalent enzyme. The protein, which crystallizes with one molecule in the asymmetric unit, consists of two subunits of identical chemical sequences, related by an almost exact binary axis. The tertiary structure of the subunits is similar to that of the pancreatic enzyme, which shows similar catalytic properties. The refinement was carried out using the restrained least-squares procedure both in the reciprocal and real spaces. The assemblage of the subunits in the dimer is described and discussed.     

Selective reduction of seminal ribonuclease by glutathione

Incubation of seminal ribonuclease with glutathione leads to the formation of a monomeric species which exhibits twice the specific activity of the native dimer. The monomer was found to possess two mixed disulfides of glutathione at residues 31 and 32, the residues ordinarily involved in the intermolecular disulfide bonds linking the subunits of the native dimer. Formation of the monomer results in only minor changes in the far ultraviolet circular dichroism spectra. The rate of the glutathione-facilitated dissociation reaction is fairly slow, requiring 60 min for completion. Attempts to dimerize the monomer all failed, implying that the dissociation reaction is irreversible. The glutathione reduced monomer was compared with the monomer formed during the regeneration of reduced, denatured bovine seminal ribonuclease in the presence of glutathione. By all criteria examined, the two monomeric forms are identical. It is concluded that the mixed disulfide monomer is the favored form of the enzyme in the presence of glutathione.     

Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease

The molecular basis of the high reactivity toward reducing agents of intersubunit disulfides at positions 31 and 32 of dimeric bovine seminal ribonuclease was investigated by studying in the monomeric enzyme the fast reaction kinetics with disulfides of the adjacent cysteine-31 and -32, exposed by selective reduction of the intersubunit disulfides. Negatively charged and neutral disulfide reagents were used for measuring the thiol reaction rates at neutral pH. The kinetics studied as a function of pH permitted us to define pK values for the thiols of interest and indicated the possibility of determining pK values of SH groups in proteins indirectly by measuring the kinetics of reactivity of the SH groups with a disulfide reagent. The results were compared with those obtained under identical conditions with synthetic thiol peptides and model compounds. The data indicate that the superreactivity of intersubunit disulfides of seminal ribonuclease is matched by the high reactivity at neutral pH of adjacent cysteine residues 31 and 32, as compared to all small thiol compounds tested. The synthetic hexapeptide segment of seminal ribonuclease Ac-Met-Cys-Cys-Arg-Lys-Met-OH, which includes the two cysteine residues of interest, was even more reactive. These data, and the other results reported in this paper, led to the conclusion that the superreactivity at neutral pH of cysteine residues at positions 31 and 32 of bovine seminal ribonuclease is primarily dependent on the nearby presence of positively charged groups, particularly the epsilon-NH2 of lysine-34, and is influenced by the adjacency of the two thiols and by the protein tertiary structure.     

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.