Formation, Structure, and Dissociation of the Ribonuclease S Three-dimensional Domain-swapped Dimer .
2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês
10.1074/jbc.m510491200
ISSN1083-351X
AutoresJorge P. López‐Alonso, Marta Bruix, Josep Font, Marc Ribó, María Vilanova, Manuel Rico, Giovanni Gotte, Massimo Libonati, Carlos González, Douglas V. Laurents,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoPost-translational events, such as proteolysis, are believed to play essential roles in amyloid formation in vivo. Ribonuclease A forms oligomers by the three-dimensional domain-swapping mechanism. Here, we demonstrate the ability of ribonuclease S, a proteolytically cleaved form of ribonuclease A, to oligomerize efficiently. This unexpected capacity has been investigated to study the effect of proteolysis on oligomerization and amyloid formation. The yield of the RNase S dimer was found to be significantly higher than that of RNase A dimers, which suggests that proteolysis can activate oligomerization via the three-dimensional domain-swapping mechanism. Characterization by chromatography, enzymatic assays, and NMR spectroscopy indicate that the structure of the RNase S dimer is similar to that of the RNase A C-dimer. The RNase S dimer dissociates much more readily than the RNase A C-dimer does. By measuring the dissociation rate as a function of temperature, the activation enthalpy and entropy for RNase S dimer dissociation were found to resemble those for the release of the small fragment (S-peptide) from monomeric RNase S. Excess S-peptide strongly slows RNase S dimer dissociation. These results strongly suggest that S-peptide release is the rate-limiting step of RNase S dimer dissociation. Post-translational events, such as proteolysis, are believed to play essential roles in amyloid formation in vivo. Ribonuclease A forms oligomers by the three-dimensional domain-swapping mechanism. Here, we demonstrate the ability of ribonuclease S, a proteolytically cleaved form of ribonuclease A, to oligomerize efficiently. This unexpected capacity has been investigated to study the effect of proteolysis on oligomerization and amyloid formation. The yield of the RNase S dimer was found to be significantly higher than that of RNase A dimers, which suggests that proteolysis can activate oligomerization via the three-dimensional domain-swapping mechanism. Characterization by chromatography, enzymatic assays, and NMR spectroscopy indicate that the structure of the RNase S dimer is similar to that of the RNase A C-dimer. The RNase S dimer dissociates much more readily than the RNase A C-dimer does. By measuring the dissociation rate as a function of temperature, the activation enthalpy and entropy for RNase S dimer dissociation were found to resemble those for the release of the small fragment (S-peptide) from monomeric RNase S. Excess S-peptide strongly slows RNase S dimer dissociation. These results strongly suggest that S-peptide release is the rate-limiting step of RNase S dimer dissociation. Formation, structure, and dissociation of the ribonuclease S three-dimensional domain-swapped dimer. VOLUME 281 (2006) PAGES 9400-9406Journal of Biological ChemistryVol. 282Issue 17PreviewPAGE 9400: Full-Text PDF Open Access Three-dimensional domain swapping is a common mechanism for oligomerization with important implications for protein evolution and amyloid formation (1Bennett M.J. Schlunegger M.P. Eisenberg D. Protein Sci. 1995; 4: 2455-2468Crossref PubMed Scopus (688) Google Scholar). In this mechanism, two monomers trade structural motifs, called "swap domains," which adopt essentially identical conformations in the monomeric and oligomeric forms; for recent reviews see Refs. 2Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Crossref PubMed Scopus (594) Google Scholar and 3Rousseau F. Schymkowitz J.W. Itzhaki L.S. Structure (Camb.). 2003; 11: 243-251Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar. The swap "domains" can range in size from a single α-helix or β-strand to large protein domains. Domain-swapped oligomers can be stable or metastable relative to their monomers. In the latter case, destabilizing conditions and high protein concentrations typically favor oligomerization by this mechanism. Bovine pancreatic ribonuclease A (RNase A) was the first protein whose oligomerization was proposed to occur by three-dimensional domain swapping (4Crestfield A.M. Stein W.H. Moore S. Arch. Biochem. Biophys. 1962; 1 (suppl.): 217-222PubMed Google Scholar). In recent years, investigation of RNase A oligomerization has shown that this protein can form a variety of dimers, trimers, and higher oligomers (5Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar) via three-dimensional domain swapping of the N-terminal α-helix (called N-dimer), the C-terminal β-strand (named C-dimer), or both (6Liu Y. Hart P.J. Schlunegger M.P. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3437-3442Crossref PubMed Scopus (184) Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Crossref PubMed Scopus (267) Google Scholar, 8Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Crossref PubMed Scopus (116) Google Scholar, 9Gotte G. Libonati M. J. Biol. Chem. 2004; 279: 36670-36679Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) (see supplemental Fig. 1 for ribbon drawings of their crystal structures). The oligomerization of RNase A has been reviewed recently (10Libonati M. Gotte G. Biochem. J. 2004; 380: 311-327Crossref PubMed Scopus (91) Google Scholar). The observation of a new two-stranded β-sheet with an amyloid-like conformation present in the C-dimer of RNase A led Eisenberg and co-workers (7Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Crossref PubMed Scopus (267) Google Scholar) to propose a model for amyloid formation based on three-dimensional domain swapping. This hypothesis has been supported by the structural characterization of domain-swapped oligomers of amyloid-forming proteins (11Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Crossref PubMed Scopus (460) Google Scholar, 12Janowski R. Kozak M. Jankowska E. Grzonka Z. Grubb A. Abrahamson M. Jaskolski M. Nat. Struct. Biol. 2001; 8: 16-20Crossref PubMed Scopus (349) Google Scholar) and is now essentially confirmed by very recent work from Eisenberg's laboratory showing that an RNase A variant with a polyglutamine insertion into the C-terminal hinge loop forms amyloid fibrils by three-dimensional domain swapping (13Sambashivan S. Liu Y. Sawaya M.R. Gingery M. Eisenberg D. Nature. 2005; 437: 266-269Crossref PubMed Scopus (230) Google Scholar). RNase A oligomerization can be induced by lyophilization from 40% acetic acid (4Crestfield A.M. Stein W.H. Moore S. Arch. Biochem. Biophys. 1962; 1 (suppl.): 217-222PubMed Google Scholar) or incubation in heated alcohol/water solutions (14Gotte G. Vottariello F. Libonati M. J. Biol. Chem. 2003; 278: 10763-10769Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Moreover, Park and Raines (15Park C. Raines R.T. Prot. Sci. 2000; 9: 2026-2033Crossref PubMed Scopus (64) Google Scholar) have shown that RNase A can dimerize in heated water without cosolvents, which suggests the existence of small amounts of dimeric RNase A in vivo. All RNase A oligomers formed are metastable and slowly dissociate into monomers. The swap domains, which consist of residues 1–15 in the N-dimer and 116–124 in the C-dimer, have the same secondary and tertiary structures regardless of whether they are bound to their own protein core in the monomer or to the alternate core in the oligomer. In contrast, their respective hinge loops, formed by residues 16–22 in the N-dimer and 112–115 in the C-dimer adopt strikingly different conformations in the dimers versus the monomer. RNase A is cleaved at the peptide bond between residues 20 and 21 by subtilisin (16Richards F.M. Vithayathil P.J. J. Biol. Chem. 1959; 234: 1459-1465Abstract Full Text PDF PubMed Google Scholar). The small fragment (residues 1–20, named S-peptide) and the large one (residues 21–124, S-protein) remain tightly associated by non-covalent interactions. This complex, RNase S, conserves the catalytic activity (16Richards F.M. Vithayathil P.J. J. Biol. Chem. 1959; 234: 1459-1465Abstract Full Text PDF PubMed Google Scholar) and native conformation (17Wyckoff H.W. Tsernoglou D. Hanson A.W. Knox J.R. Lee B. Richards F.M. J. Biol. Chem. 1970; 245: 305-328Abstract Full Text PDF PubMed Google Scholar, 18Rico M. Bruix M. Santoro J. González C. Neira J.L. Nieto J.L. Herranz J. Eur. J. Biochem. 1989; 183: 623-638Crossref PubMed Scopus (65) Google Scholar, 19Kim E.E. Varadarajan R. Wyckoff H.W. Richards F.M. Biochemistry. 1992; 31: 12304-12314Crossref PubMed Scopus (122) Google Scholar) of uncleaved RNase A but shows a reduced conformational stability (20Catanzano F. Giancola C. Graziano G. Barone G. Biochemistry. 1996; 35: 13378-13385Crossref PubMed Scopus (32) Google Scholar, 21Neira J.L. Sevilla P. Menéndez M. Bruix M. Rico M. J. Mol. Biol. 1999; 285: 627-643Crossref PubMed Scopus (69) Google Scholar, 22Chakshusmathi G. Ratnaparkhi G.S. Mudhu P.K. Varadarajan R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7899-7904Crossref PubMed Scopus (20) Google Scholar). Although N-terminal domain swapping cannot occur, RNase S monomers might still be able to oligomerize by swapping C termini, which are not cut by subtilisin. However, given the reduced conformational stability of RNase S, many would not expect it to form oligomers. Here, we describe the discovery of RNase S dimers; this is the first reported case, to our knowledge, of a two-chain protein that oligomerizes via the three-dimensional domain-swapping mechanism. The main objectives of this present work are to (i) compare the ability of RNase S to form domain-swapped oligomers relative to RNase A, (ii) characterize the structure of the RNase S dimer, and (iii) measure the kinetics and thermodynamics of RNase S dimer dissociation and propose a mechanism for this process. Materials—Bovine pancreatic ribonuclease A (grade XII-A, lot 104H-7110) was obtained from Sigma and further purified by cation-exchange chromatography as described (5Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar) prior to use. Bovine pancreatic ribonuclease S (grade XII-S, lot 106F-8055), S-peptide (grade XII-PE, lot 99F8205), and S-protein (grade XII-PR, lot 125C8045) also obtained from Sigma, eluted as single peaks from a cation exchange column and were used as supplied. Recombinant RNase A uniformly labeled with 15N or 15N and 13C was prepared as described previously (23Ribó M. Benito A. Canals A. Nogués M.V. Cuchillo C.M. Vilanova M. Methods Enzymol. 2001; 341: 221-234Crossref PubMed Scopus (26) Google Scholar) using labeled Martek9 medium from Spectra Stable Isotopes. Na2HPO4 and NaH2PO4 were obtained from Merck and combined in equal molar proportions to give a solution that is 0.20 m in total Pi and has a pH of 6.7. Double-distilled water was deionized using a MilliQ system. All other reagents were the highest purity grade available. Oligomer Formation—The ability of RNase S and S-protein to form oligomers was tested using two procedures that efficiently induce oligomerization of RNase A; namely (i) incubation in 40% glacial acetic acid, 60% water for 1 h, followed by lyophilization (4Crestfield A.M. Stein W.H. Moore S. Arch. Biochem. Biophys. 1962; 1 (suppl.): 217-222PubMed Google Scholar) (HAc/lyophilization) and (ii) high temperature incubation in 40% ethanol, 60% water at high protein concentrations (50–200 mg/ml) (EtOH/heat) (14Gotte G. Vottariello F. Libonati M. J. Biol. Chem. 2003; 278: 10763-10769Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Incubation temperatures ranging from 30 to 60 °C were tested. NaPi buffer (0.2 m, pH 6.7) was added to the samples after lyophilization or heat treatment. Pi binds to His12 and His119 at the active site (24Cohen J.S. Griffen J.H. Schechter A.N. J. Biol. Chem. 1973; 248: 4305-4310Abstract Full Text PDF PubMed Google Scholar); because these residues are contributed by different monomers, Pi binding stabilizes domain-swapped oligomers of RNase as does the binding of DNA (25Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Crossref PubMed Scopus (41) Google Scholar). Chromatography—All chromatography experiments were performed on an Amersham Biosciences ΔKTA FPLC system at ambient temperature (18–20 °C) using Mono S 5/5 and Superdex HR 75 10/30 columns for cation exchange and gel filtration, respectively. Cation exchange chromatography was carried out by applying a linear gradient of 20–200 mm NaPi (pH 6.70) as previously described (14Gotte G. Vottariello F. Libonati M. J. Biol. Chem. 2003; 278: 10763-10769Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Gel filtration was performed in 0.2 m NaPi buffer. Elution volume determination and peak integration was performed using the Amersham Biosciences Unicorn software. Thermal Denaturation—Thermal transitions (0–70 °C) of RNase S and RNase A (≈0.5 mg/ml) in 40% ethanol, 60% water were recorded in a 0.1-cm cuvette using a JASCO J-810 circular dichroism spectrometer equipped with a Peltier temperature control unit. These samples were unbuffered, pH ≈ 6.8, to be consistent with the EtOH/heat oligomerization conditions. The scan speed was 60 °C/h. The reversibility of unfolding was checked by recording the ellipticity during recooling to 0 °C and was found to be better than 95%. The data shown are representative of four independent experiments. Enzyme Kinetics—The enzymatic activities of ribonuclease monomers and dimers were measured against single stranded yeast RNA (ssRNA) 4The abbreviations used are: ssRNA, single stranded RNA; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; MD, molecular dynamics; MOPS, 4-morpholinepropanesulfonic acid. using the Kunitz assay (26Kunitz M. J. Biol. Chem. 1946; 164: 563-568Abstract Full Text PDF PubMed Google Scholar) and against synthetic double stranded RNA, poly(A)·poly(U) as previously described (27Libonati M. Sorrentino S. Methods Enzymol. 2001; 341: 234-248Crossref PubMed Scopus (46) Google Scholar). All assays were repeated in duplicate, and the uncertainty of these activity values is typically ∼±7% and ±15%, against ssRNA and poly(A)·poly(U), respectively (5Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar). Because gel filtration analysis found that the RNase S dimer sample contained 56% dimer and 44% monomer, the activity of 100% dimer was calculated by subtracting the activity due to monomer from the observed activity and then dividing by the fraction of dimer. The specific activity values were then normalized to the RNase A monomer. Nuclear Magnetic Resonance—All NMR spectra were recorded at 25.0 °C, in NaPi buffer (0.2 m, pH 6.7) containing 90% H2O, 10% D2O or in 99.9% D2O using the most upfield resonance of sodium 4,4-dimethyl-4-silapentane-1-sulfonate as the internal chemical shift reference on a Bruker 800 MHz Avance US2 NMR spectrometer equipped with a triple resonance (1H,13C,15N) probe and X, Y, Z-gradients. Three-dimensional 13C,15N,1H NHCOCA, NHCA, and NHCACB spectra were recorded on a monomeric RNase A sample uniformly labeled in 13C and 15N. Peak assignments were made by following standard procedures in combination with comparison of previous assignments (18Rico M. Bruix M. Santoro J. González C. Neira J.L. Nieto J.L. Herranz J. Eur. J. Biochem. 1989; 183: 623-638Crossref PubMed Scopus (65) Google Scholar, 21Neira J.L. Sevilla P. Menéndez M. Bruix M. Rico M. J. Mol. Biol. 1999; 285: 627-643Crossref PubMed Scopus (69) Google Scholar). Based on these assignments, two-dimensional 1H NOESY (mixing time = 50 ms), COSY, and total correlation spectroscopy spectra of monomeric and dimeric RNase S were recorded and assigned in the same solution conditions and temperature. The assignments are deposited in the BMRB data bank. RNase S dimer samples for NMR contained an excess of S-peptide to slow dissociation (see below). Structural Modeling of the RNase S Dimer—The chemical shift and NOE data sets of RNase A, RNase S, and the RNase S dimer are almost identical; significant differences are only observed near the subtilisin cleavage site (see Fig. 2 in Neira et al. (21Neira J.L. Sevilla P. Menéndez M. Bruix M. Rico M. J. Mol. Biol. 1999; 285: 627-643Crossref PubMed Scopus (69) Google Scholar)) and in the C-terminal hinge loop (see Fig. 2, above). Therefore, their tertiary structures are very similar except at those sites. Starting from two RNase A solution structures, the RNase S dimer structure was modeled by cleaving the 20–21 peptide bonds and then changing the backbone torsion angles of the C-terminal hinge loop to a C-dimer-like conformation using the program Sybyl (Tripos, Inc.). This model was refined with extensive (500 ps) MD simulations with explicit solvent. For further details, see Ref. 28Gómez-Pinto I. Cubero E. Kalko S.G. Monaco V. van der Marel G. van Boom J.H. Orozco M. González C. J. Biol. Chem. 2004; 279: 24552-24560Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar and the supplemental information. Dimer Dissociation—The kinetics of the separation of RNase S dimers, in NaPi buffer (0.2 m, pH 6.7), into monomers was monitored by gel filtration analysis of aliquots incubated at 0, 19.0, 25.0, 31.2, and 37.0 °C. To determine the dissociation rate constant, k, a least-squares algorithm was used to fit first order kinetic rate equations to the experimentally observed increase in monomer (M) or the decrease in dimer (D) with time (t), D(t)=D(t=0)exp-kt+D(t=∞)(Eq. 1) and, M(t)=(1-M(t=0))exp-kt+M(t=∞)(Eq. 2) The rate constants (k) as a function of the inverse of the absolute temperature (T) were analyzed using the linear form of the Arrehenius equation, lnk=-ΔH‡/RT+ΔS‡/R(Eq. 3) to determine the apparent activation enthalpy (ΔH‡) and entropy (ΔS‡) for rate-limiting step of RNase S dimer dissociation. HAc/Lyophilization-induced Oligomerization—To test whether or not RNase S can form three-dimensional domain-swapped oligomers under the conditions that induce oligomerization of RNase A, samples of both proteins were subjected to HAc/lyophilization treatment, and the subsequent products were separated by chromatography (Fig. 1). Cation exchange chromatography was tried first, because it highly resolves RNase A oligomers (5Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar). Here, a very good separation of RNase A oligomers was also obtained with a 0.02–0.20 m NaPi gradient, and the elution positions and oligomer yields are consistent with previous results (5Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar, 10Libonati M. Gotte G. Biochem. J. 2004; 380: 311-327Crossref PubMed Scopus (91) Google Scholar) (Fig. 1a). However, in the case of HAc/lyophilization treated RNase S, a single peak with a long trailing shoulder was observed to elute soon after the main monomeric peak. Next, the separation of the oligomerization products was attempted using a gel filtration column equilibrated in 200 mm NaPi. As previously observed (5Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Crossref PubMed Scopus (75) Google Scholar), RNase A tetramers elute earliest, followed by trimers, the C-dimer, the N-dimer, and finally the monomer (Fig. 1b). The ability to partly resolve the two dimers of RNase A by gel filtration is likely because of the more elongated shape of the C-dimer (Fig. 1b, inset). The chromatogram of RNase S, oligomerized by the HAc/lyophilization procedure, reveals the presence of two peaks, which elute before monomeric RNase S and that were not observed in untreated RNase S (Fig. 1b). The smaller peak, whose yield is ∼5 ± 1% (1σ, n = 11), has an elution volume that corresponds approximately to a trimeric species. The major RNase S oligomer is probably a dimer, as it elutes 2.08 ± 0.02 ml (1σ, n = 32) before monomeric RNase S, whereas the RNase A N-dimer and C-dimer elute 1.90 ± 0.02 ml and 2.15 ± 0.01 mσ (1 s, n = 4), respectively, before the RNase A monomer. This putative dimer is referred to henceforth as the RNase S dimer. It is remarkable that the yield of the RNase S dimer (32 ± 3%, 1σ, n = 11) is significantly higher than that of the RNase A C-dimer (18 ± 1%) (29Gotte G. Libonati M. Laurents D.V. J. Biol. Chem. 2003; 278: 46241-46251Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) and is even marginally superior to the typical yields (26–30%) (10Libonati M. Gotte G. Biochem. J. 2004; 380: 311-327Crossref PubMed Scopus (91) Google Scholar) of the sum of all the RNase A oligomers formed after HAc/lyophilization treatment. The stability of the interactions maintaining the quaternary structure of the RNase S oligomers could be low. Phosphate binding to the active site of RNase oligomers cross-links the subunits and thereby stabilizes them. RNase S oligomers might have been present but dissociated in the early stages of cation exchange chromatography, where the concentration of Pi is minimal. To test this possibility, the products of RNase S oligomerization were applied to gel filtration column equilibrated with 200, 90, or 10 mm NaPi buffer (Fig. 1c). The recovery of oligomers decreases substantially as the Pi concentration is lowered. S-protein might be able to oligomerize because it contains the C-terminal swap domain of RNase A. HAc/lyophilization-treated S-protein analyzed by gel filtration yielded a broad peak whose elution volume is close to that of the RNase S monomer (Fig. 1d). The shoulder preceding the elution of the main peak is more prominent for the HAc/lyophilization-treated S-protein than for untreated S-protein; this shoulder might possibly be because of S-protein oligomers, which dissociate into monomers during the chromatography run. A strikingly different result is obtained, however, if a 1.3 molar excess of S-peptide is included in the NaPi buffer used to redissolve the lyophilized S-protein powder; namely, significant amounts of oligomers are detected (Fig. 1d). These results strongly suggest that S-protein can oligomerize by three-dimensional domain swapping of the C-terminal β-strand, as does RNase A, but that S-protein oligomers are much less stable. Formation of RNase S Oligomers in Heated 40% EtOH—Oligomer formation by RNase S was also found to occur when the protein was incubated at elevated temperatures in 40% ethanol. The average percent oligomer yields as a function of the incubation temperature are shown in Fig. 2a. The highest yields of oligomers are obtained near 40 °C for RNase S but at a much higher temperature, ∼60 °C, for RNase A (14Gotte G. Vottariello F. Libonati M. J. Biol. Chem. 2003; 278: 10763-10769Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). To gain further insight, the thermal denaturation in 40% ethanol of RNase A and S, the concentration of the latter being 43 μm, was followed by CD and thermal midpoint (TM) values of 30.7 ± 0.2 °C for RNase S and 42.0 ± 0.2 °C for RNase A were obtained (Fig. 2b). The TM value for RNase S is concentration-dependent; when corrected for its concentration under oligomerization conditions (7.3 mm) using the expression of Catanzano et al. (20Catanzano F. Giancola C. Graziano G. Barone G. Biochemistry. 1996; 35: 13378-13385Crossref PubMed Scopus (32) Google Scholar) increases to ≈41 °C (see the supplemental text for additional data analysis details). Altogether, these data suggest that RNase A oligomerizes most efficiently at high temperatures where it is completely denatured, whereas RNase S oligomerization is highest at physiological temperatures near the midpoint of its unfolding transition. Enzymatic Activities—Because the catalytic activities of RNase A depend strictly on its tertiary and quaternary structure (10Libonati M. Gotte G. Biochem. J. 2004; 380: 311-327Crossref PubMed Scopus (91) Google Scholar), enzymatic assays can shed light on the nature of the RNase S dimer structure. First, both the monomeric and dimeric species of RNase A and RNase S were assayed against yeast ssRNA. The RNase S dimer shows significant activity against ssRNA, which parallels that shown by the RNase A C-dimer (data not shown) and constitutes good evidence that the subunits of the dimer have correctly folded tertiary structures. The RNase A C-dimer shows a remarkably increased activity against poly(A)·poly(U) relative to the monomer, whereas the enhanced activity of the N-dimer against poly(A)·poly(U) is much smaller (9Gotte G. Libonati M. J. Biol. Chem. 2004; 279: 36670-36679Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30Sorrentino S. Barone G. Bucci E. Gotte G. Russo N. Libonati M. D'Alessio G. FEBS Lett. 2000; 466: 35-39Crossref PubMed Scopus (26) Google Scholar). Against poly(A)·poly(U), the RNase S dimer demonstrated a strongly enhanced activity like that of the RNase A C-dimer when assayed side-by-side (Fig. 3a). Structural Characterization by NMR—The aromatic region of the two-dimensional NOESY 1H spectra of the RNase S monomer and dimer are shown in Fig. 3b. Although the chemical shifts of most peaks are essentially identical, those of the RNase S dimer are broader, which reflects its slower tumbling in solution. The ring protons of Tyr115, a residue located in the C-terminal hinge loop, alter their chemical shifts in the RNase S dimer relative to the monomer (Fig. 3b). The Tyr115 1Hδs and Cys58 1Hβs have strong NOE signals in the monomeric forms of RNase S and A, which are absent in the spectra of the RNase S dimer (Fig. 3b) as well as the C-dimer of RNase A (data not shown). The differences in the backbone chemical shifts of monomeric versus dimeric RNase S are small, except in the hinge loop region where significant differences are observed (Fig. 3c). As NMR chemical shifts are exquisitely sensitive to conformation, these data indicate that the backbone structure of the monomer and dimer are similar, except in the hinge loop region. A key conformational difference in the hinge loop is revealed by the NOESY spectroscopy. Strong Asn113 1Hα-Pro114 1H,H′δ NOE signals, which are diagnostic of a trans peptide bond, are observed in the RNase S dimer spectrum but are absent in the monomer spectrum (Fig. 3d). Similar NOEs are observed for the RNase A C-dimer but are absent in RNase A monomer and N-dimer NOESY spectra (data not shown). The NMR signals that are characteristic for C-terminal domain swapping, namely those of the Tyr115 side chain and NOE signals between the Asn113 1Hα-Pro114 1H,H′δ, are lost when the RNase S dimer dissociates (see the supplemental text describing additional NMR experiments). Hydrogen Exchange—Some 30 amide protons in the RNase S dimer resist exchange for more than 2.5 h at pH 6.7, 25 °C when the protein is dissolved in D2O but are fully exchanged after 14 h. Based on these observations, protection factors on the order of 105-106 can be estimated for these protons. These values are comparable to those previously reported for the RNase S monomer (21Neira J.L. Sevilla P. Menéndez M. Bruix M. Rico M. J. Mol. Biol. 1999; 285: 627-643Crossref PubMed Scopus (69) Google Scholar, 22Chakshusmathi G. Ratnaparkhi G.S. Mudhu P.K. Varadarajan R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7899-7904Crossref PubMed Scopus (20) Google Scholar), and this resemblance suggests that the conformational stabilities of the RNase S monomer and dimer are also similar. The identities of the highly protected protons match closely in the RNase S monomer and dimer; this is an additional indication that they share similar tertiary structures (see supplemental Table 1 for a list of the protected amide protons in the monomer and dimer). Structural Model—A structure of the RNase S dimer modeled on the basis of the NMR data is shown in Fig. 3e. Overall, the structure is very similar to the C-dimer of RNase A (7Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Crossref PubMed Scopus (267) Google Scholar), cleaved between Ala20 and Ser21 in the two subunits. The evolution of the structure during the course of the MD simulation (shown in supplemental Fig. 2) suggests that it is well folded and contains no strained regions. Dissociation Kinetics—The stability of the RNase S dimers against dissociation upon incubation at various temperatures was monitored by gel filtration chromatography, and the results are shown in Fig. 4a. Essentially equivalent rate constants were found by fitting the increase in monomer or the decrease in dimer with time (data not shown). The dissociation kinetics depend strongly on temperature, with the kinetic lifetimes at 0 and 37 °C being: τ0° = 78,000 ± 1500 min (54 ± 1 days) and τ37° = 5 ± 1.5 min, respectively. In comparison, the monomerization of the RNase A C-dimer is very slow under these conditions. After 1 month at 37 °C in 0.20 m NaPi, pH 6.7, less than 10% had dissociated into monomers (data not shown). The ability of additional S-peptide to stabilize the RNase S dimers was also tested. When a 3-fold molar excess of S-peptide was added to RNase S dimers, their dissociation kinetics slowed dramatically. At 31.2 °C with a 3-fold excess of S-peptide, less than 25% of the RNase S dimer dissociates after 3 h, whereas without additional S-peptide, the dimer has essentially completely disassociated after 2 h (Fig. 4a). At 37 °C, a 3-fold molar excess of S-peptide reduces the RNase S dissociation rate 20-fold, with τ37°, 3× S-pep = 106 ± 18 min. Thermodynamics of RNase S Dimer Dissociation—Arrhenius plots of the rate of RNase S dimer dissociation and the release of S-peptide from monomeric RNase S determined by Goldberg and Baldwin (31Goldberg J.M. Baldwin R.L. Biochemistry. 1998; 37: 2556-2563Crossref PubMed Scopus (42) Google Scholar) are
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