Local Folding of the N-terminal Domain of Escherichia coli RecA Controls Protein-Protein Interaction

To obtain structural information about the self-association of the protein RecA, we studied urea denaturation of RecA by circular dichroism spectroscopy and gel filtration. Gel filtration analysis showed that urea at low concentrations, 1.0–1.2m, dissociated the RecA oligomer to almost a monomeric state prior to the unfolding of each molecule. Upon treatment with 1.0 m urea, the circular dichroism spectrum showed a decrease in the α-helical content of RecA. A similar decrease was observed in the absence of urea for RecA at an extremely low protein concentration; the RecA oligomer dissociated to an almost completely monomeric state. The properties of RecA at low urea concentrations were similar to those of a truncated RecA lacking the first 33 N-terminal residues (Δ33RecA). Addition of a synthetic peptide corresponding to the 33 N-terminal residues to Δ33RecA increased the α-helical content. These results suggest that local folding of the N-terminal domain is coupled to protein-protein interactions of monomeric RecA, which are involved in the regulation of filament formation. The dissociation constant for interaction between RecA monomers was determined from the ellipticity data to be 0.1 μm. To obtain structural information about the self-association of the protein RecA, we studied urea denaturation of RecA by circular dichroism spectroscopy and gel filtration. Gel filtration analysis showed that urea at low concentrations, 1.0–1.2m, dissociated the RecA oligomer to almost a monomeric state prior to the unfolding of each molecule. Upon treatment with 1.0 m urea, the circular dichroism spectrum showed a decrease in the α-helical content of RecA. A similar decrease was observed in the absence of urea for RecA at an extremely low protein concentration; the RecA oligomer dissociated to an almost completely monomeric state. The properties of RecA at low urea concentrations were similar to those of a truncated RecA lacking the first 33 N-terminal residues (Δ33RecA). Addition of a synthetic peptide corresponding to the 33 N-terminal residues to Δ33RecA increased the α-helical content. These results suggest that local folding of the N-terminal domain is coupled to protein-protein interactions of monomeric RecA, which are involved in the regulation of filament formation. The dissociation constant for interaction between RecA monomers was determined from the ellipticity data to be 0.1 μm. RecA, a protein from Escherichia coli, is a small protein with a relative molecular mass of approximately 38,000 (1Horii T. Ogawa T. Ogawa H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 313-317Crossref PubMed Scopus (194) Google Scholar, 2Sancar A. Stachelek C. Konigsberg W. Rupp W.D. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2611-2615Crossref PubMed Scopus (202) Google Scholar), characterized by numerous functional properties in its active form (3Cox M.M. Lehman I.R. Annu. Rev. Biochem. 1987; 56: 229-262Crossref PubMed Scopus (219) Google Scholar, 4Egelman E.H. Curr. Opin. Struct. Biol. 1993; 3: 189-197Crossref Scopus (63) Google Scholar, 5Roca A.I. Cox M.M. Crit. Rev. Biochem. Mol. Biol. 1990; 25: 415-456Crossref PubMed Scopus (363) Google Scholar). These actions include homologous recombination of DNA strands, self-polymerization in the presence or absence of DNA, DNA-dependent hydrolysis of ATP, and stimulation of autocleavage of LexA and certain other repressor proteins. RecA forms various quaternary structures, which are inherent in the activities exhibited by the protein. RecA is active only when it forms a nucleoprotein filament, which consists of a helical array of monomers bound to single-stranded DNA (ssDNA). 1The abbreviations used are: ssDNA, single-stranded DNA; ATPγS, adenosine 5′-O-(3-thiotriphosphate); HPLC, high performance liquid chromatography. This highly ordered structure is sustained predominantly by protein-protein interactions between contiguous monomers (6Williams R.C. Spengler S.J. J. Mol. Biol. 1986; 187: 109-118Crossref PubMed Scopus (81) Google Scholar). RecA is an α/β protein, and its polypeptide chain is folded into three continuous domains, referred to as the N-terminal, central, and C-terminal domains (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). The nucleotide-binding site has been assigned to the central domain by analyzing the structure of the crystal containing ADP (8Story R.M. Steitz T.A. Nature. 1992; 355: 374-376Crossref PubMed Scopus (560) Google Scholar). Although no crystal of the RecA-DNA complex has been analyzed, two unordered loops in the central domain are presumed to be the DNA-binding sites (9Wang Y. Adzuma K. Biochemistry. 1996; 35: 3563-3571Crossref PubMed Scopus (48) Google Scholar, 10Voloshin O.N. Wang L. Camerini-Otero R.D. Science. 1996; 272: 868-872Crossref PubMed Scopus (73) Google Scholar, 11Malkov V.A. Camerini-Otero R.D. J. Biol. Chem. 1995; 270: 30230-30233Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12Gardner R.V. Voloshin O.N. Camerini-Otero R.D. Eur. J. Biochem. 1995; 233: 419-425Crossref PubMed Scopus (25) Google Scholar, 13Maraboeuf F. Voloshin O. Camerini-Otero R.D. Takahashi M. J. Biol. Chem. 1995; 270: 30927-30932Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). One interesting feature of crystalline RecA is that the RecA molecules form a helical filament even in the absence of DNA (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). Based on the structure in the crystal, two regions involved in protein-protein interaction have been identified; the N-terminal domain interacts with a complementary site in the central domain of the adjacent molecule in the filament. Nevertheless, the filament structure in the crystal resembles that of an inactive filament when observed by electron microscopy (14Stasiak A. Egelman E.H. Experientia (Basel). 1994; 50: 192-203Crossref PubMed Scopus (67) Google Scholar). Additionally, in the absence of DNA and nucleotides, RecA exists in multiple oligomeric states (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar, 16Brenner S.L. Zlotnick A. Stafford III, W.F. J. Mol. Biol. 1990; 216: 949-964Crossref PubMed Scopus (42) Google Scholar) and protein-protein interactions within these oligomers have not been well studied. We have recently studied the properties of an N-terminally truncatedE. coli RecA, designated Δ33RecA, which lacks the first 33 amino acid residues (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). Δ33RecA shows reduced self-association, which results in reduced DNA binding and ATP hydrolysis. By contrast, in the presence of MgCl2 Δ33RecA does self-associate, although a higher protein concentration and longer time are required for completion of the process than for the wild-type protein. These results suggested that the 33 N-terminal amino acid residues play an important role not only in protein-protein interactions but also kinetic regulation of the self-association process. Separately, a controversial observation has been reported; a synthetic peptide corresponding to the first 24 amino acids of RecA was able to bind to DNA when the peptide underwent a conformational from an unordered structure to an A. S.L. J. Mol. Biol. 1988; Scopus (25) Google Scholar). is required to the involved in the self-association of RecA. To the self-association process in we studied the denaturation of RecA. the of an oligomeric protein RecA, denaturation information about protein-protein it dissociation of the oligomers in to unfolding of the for a and protein RecA, denaturation in the of its functional we that unfolding was coupled to dissociation of the RecA the properties of RecA at low urea concentrations were similar to those of We a peptide corresponding to the first 33 N-terminal amino acid residues of RecA and its The results suggest that a local folding in the N-terminal domain protein-protein interaction of monomeric RecA. coli RecA S. K. H. T. Ogawa T. Ogawa H. Biochemistry. PubMed Scopus Google Scholar) and Δ33RecA (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar) were as The RecA concentration was determined an of S. K. H. T. Ogawa T. Ogawa H. Biochemistry. PubMed Scopus Google Scholar). A corresponding to 33 N-terminal residues of coli RecA, was a peptide by the The of the peptide was The peptide was by on a a of in The peptide was at The of the peptide was by amino acid analysis and N-terminal The peptide concentration was determined by amino acid and adenosine were from and other of were were at on a with an and denaturation protein were for at at each urea concentration prior to Gel filtration analysis was a in a denaturation the were at for 24 at each concentration of urea and were with containing the concentration of urea at a of The was the and was also used for The was by the at or the were at approximately The were at for in containing various concentrations of urea, and MgCl2 was to a concentration of the was at a the were at of ATP by RecA was by an J. Cox M.M. Biochemistry. 1986; 25: PubMed Scopus Google Scholar) with (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). We the denaturation of wild-type RecA were of the for at showed no in the of that been at for and 24 These results that the folding was within at each urea The of RecA in the are in A. urea, RecA showed at approximately and which is of an the presence of or m urea, the ellipticity at that at was almost the concentration of urea was increased to m and the the Addition of m urea in of the in the ellipticity at as the concentration of urea increased the are as the to it to the of wild-type RecA and Although were to a small was at low urea The was to be at approximately 1.0 m urea, although no state was observed and the in the was to The state at a urea concentration of approximately approximately of the This reduced that RecA in the presence of m urea was in a state. By contrast, a was not observed for Δ33RecA low concentrations of urea, to on the of its which was to that of wild-type RecA with These results that the observed for the wild-type protein with approximately 1.0 m urea is with the N-terminal The of RecA in the are in A. The in the presence of and m urea were similar to each the concentration of urea was increased to the of the spectrum and its at from to The in the ellipticity at with the of urea concentration is in This was at and the of the first was higher than that of the The was at approximately 1.0 for the first that for the was to was of that the of the from to the first This that the the of the amino acids were at approximately 1.0 m the was almost in the presence of urea, the was an ordered which from that in the state. that the for the first in the and for the in the were to each The spectrum of Δ33RecA in the state was similar to that of the wild-type protein with m urea (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). for urea denaturation was from that of wild-type no was in the of urea with the that the N-terminal is with the of the wild-type protein with approximately 1.0 m RecA exists in multiple oligomeric states (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar). To the observed or the of these on the protein concentration was the the for the first was to approximately m urea at a protein concentration of for it was approximately 1.0 m urea This that the first in the spectrum was with an oligomeric state of the protein. a protein concentration of the was almost the as that for This at concentrations the oligomeric states of RecA were to in the structure by the low concentration of A on the protein concentration was also observed for the in the a protein concentration of the was to approximately m urea, which to be to the m urea, for protein in the to the in the the protein concentration to in a of the to approximately m These results suggest that in the was to the oligomeric states of the protein than that in the This be by the that the and quaternary of the protein. it that the observed with low concentrations of urea is with the dissociation process in the denaturation of RecA. denaturation of the oligomeric dissociation to we to the observed were oligomeric or To the oligomeric state and the of the gel filtration of RecA was in the presence of urea the absence of urea, RecA at was predominantly in the of the and small of the protein were also at that RecA exists as various forms of oligomers in the concentration of urea was increased to 1.0 the and the of the RecA molecules as in the in A. This was to the process of RecA oligomer dissociation as the urea concentration the concentration of urea was increased to a the and a small was at which was the the in the of the of molecular mass in the an of to a relative molecular mass of approximately in a folded This is than that of the RecA from its amino acid a urea concentration of RecA was almost as a which was the of the the were also observed for m unfolding of the polypeptide chain is to of the of the it is to that these the to an state of the RecA molecule. The small in the of from to for urea concentrations 1.0 m also to a of the concentration of urea was increased the increased only but the of that the of the molecules a we the of Δ33RecA the absence of urea, the of Δ33RecA was a than that of the wild-type that it exists as a a urea concentration of Δ33RecA was the of the not denaturation of Δ33RecA not we that to an state of the molecule. This the that the of wild-type RecA that the to an state. the results that dissociation of RecA oligomer is by unfolding of the To the of urea on the oligomeric state of RecA, self-association in the presence of urea was by MgCl2 to the MgCl2 has been to of RecA (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar). The from RecA is to be by the of the at as in A. urea, the at increased the of MgCl2 to a concentration of a urea concentration of an in also the of and was almost The in urea and the time required for was longer m RecA, was in even at a urea concentration 1.0 as in These results suggest that the structural at approximately 1.0 m urea RecA of By contrast, when the protein concentration was increased to the even in the presence of 1.0 m and m urea The were and the time required for was than These results that the in self-association was to by the protein We also studied the of RecA to bind to ATP in the presence of urea by the in the ATP bound to RecA in the absence of urea, the at increased as in A R. Masui R. T. S. R. Kuramitsu S. J. Biochem. 1994; PubMed Scopus Google Scholar). ATP was to RecA with m urea, a in the was observed only at 1.0 m urea, the of ATP in a in ellipticity These binding of ATP to RecA the of the were than the of the for RecA and ATP m urea, in the ellipticity was almost no binding of ATP to the protein. these results suggest that the presence of urea the of RecA to bind to This the observation that the of no on the in the The of urea denaturation that the oligomeric state of RecA was in the of a of oligomeric forms on protein concentration as well as and (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar), we that the spectrum of RecA with the protein To the of the oligomeric state of RecA on its we the oligomeric state of RecA by gel filtration. The of RecA are at various concentrations from to the protein concentration the of the protein molecules These no or a the various forms of the when RecA was at the was at a of approximately corresponding to a protein with a relative molecular mass of approximately This was than the of 38,000 from its but was than for a of RecA. Although a of the RecA it that the of oligomeric states of RecA was the monomeric state at extremely low protein we the spectrum of RecA continuous This spectrum was similar to that of Δ33RecA in the ellipticity at on the protein concentration; it increased the concentration and it almost constant at the By contrast, the ellipticity at was in the between 0.1 and protein. These results that the decrease in ellipticity at approximately was to the dissociation even the dissociation was by the protein to ellipticity at be to be an of an oligomeric state of RecA. The of the oligomeric from the on protein concentration similar to that by electron microscopy (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar). an constant for the interaction between RecA monomers be from the data in although are of which on the relative of oligomers (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar). the monomers and are states in the the be as a in the dissociation constant is as in The The concentration of monomers and is to the concentrations, as in The observed ellipticity is as in and are the of the monomers and The of and were determined from the data in The was determined by to the observed at various concentrations of The determined was approximately 0.1 μm. in the The was by the RecA not the of the RecA containing 1.0 m urea to approximately of those in the absence of urea not These results suggest that to the structure of RecA by the protein concentration or by low concentrations of urea are and also to the that is with the dissociation process. The properties of RecA at low concentrations of urea were similar to those of Δ33RecA, which lacks the first 33 amino acid residues This that the unfolding dissociation of RecA oligomers in the N-terminal To the of the N-terminal in the we a peptide corresponding to residues of the wild-type protein, designated and characterized its structural The spectrum of not feature of an ordered as in Δ33RecA was to the ellipticity of the showed an in the regions at and with the of the of and The between the spectrum of the and that of Δ33RecA is in The spectrum to be of an it shows at and the spectrum of the of Δ33RecA and was similar to that of the wild-type protein, although the ellipticity of the was than that of the not These results that an were interaction between Δ33RecA and Addition of to RecA, even to the of the wild-type protein in of α-helical as in interaction between the that a interaction between and RecA the conformational was a we Δ33RecA with which is similar to RecA in approximately and and observed no in the spectrum of the with the of the not This the that with To the functional interaction of these the of binding to Δ33RecA was studied an Δ33RecA showed no as (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). Addition of even to the of Δ33RecA, no in the not an active nucleoprotein filament, that the complex to form a helical filament, which is to the self-association process. This also the of interaction between and Δ33RecA the absence of DNA and nucleotides, RecA exists in multiple oligomeric states in with the on has that and are the oligomeric S.L. Zlotnick A. Stafford III, W.F. J. Mol. Biol. 1990; 216: 949-964Crossref PubMed Scopus (42) Google Scholar). also and of (15Brenner S.L. Zlotnick A. Griffith J.D. J. Mol. Biol. 1988; 204: 959-972Crossref PubMed Scopus (86) Google Scholar). the are and and the protein-protein interactions within the be from those within the Camerini-Otero R.D. Egelman E.H. J. 1995; Full Text PDF PubMed Scopus Google Scholar). the filament structure observed in the crystalline the N-terminal domain to the between the (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). We studied Δ33RecA, a truncated RecA protein lacking the first 33 amino acid and results that the N-terminal of RecA an important role in protein-protein interaction not only in the but also in oligomeric forms (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). and Egelman Egelman E.H. Struct. Biol. PubMed Scopus Google Scholar) recently a structure for the in which the N-terminal domain is involved in protein-protein interactions but in a from that within the we the self-association process denaturation and results that unfolding and folding the dissociation of RecA oligomers and the of RecA unfolding of the RecA molecule at low concentrations of urea or at an extremely low protein concentration to a by gel filtration. is that the RecA exists in a the with m urea showed that RecA an ordered although it was from that of the state. no in the of acid was observed in the of denaturation of RecA not These results the that the state to a state. it is that unfolding dissociation in a of the molecule. The results also suggest the at which the local unfolding RecA with low concentrations of urea showed properties from the with that of the protein, the at was as was that of the protein at an extremely low concentration the spectrum was and the at was from to the binding to ATP was than that for the protein and a was observed only at RecA showed reduced self-association by but a higher protein concentration and longer time than that used urea the protein to and in the presence of these properties those of Δ33RecA (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). no was in the denaturation of for Δ33RecA in the of low urea we that a folding of the N-terminal domain protein-protein The results of the with the crystal structure (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar, Egelman E.H. Struct. Biol. PubMed Scopus Google Scholar), a of the protein-protein interaction of RecA the absence of urea, the N-terminal domain is at low protein concentrations and it into an self-association when the protein concentration is the presence of low concentrations of urea, the N-terminal domain is even at high protein concentrations, but self-association to Δ33RecA only at high protein concentrations of urea have no on Δ33RecA to of the N-terminal The unfolding of the N-terminal domain in the presence of urea, even at a high protein be to its from the molecule and also from the helical filament (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). To that the N-terminal domain a conformational self-association, we a folding interaction and Δ33RecA and an in the structure the interaction of these two Based on the it is that the site on Δ33RecA that is the in the wild-type protein, the N-terminal domain of an adjacent than the which its N-terminal domain RecA molecules are of folded and Δ33RecA but the of of those is from that of the wild-type protein. This RecA protein lacks the to form a filament which is for the spectrum of the was similar to that of Δ33RecA, and the of Δ33RecA was not by the of not the of these two to a protein of into an active filament. We that the on Δ33RecA corresponding to the N-terminal domain of the wild-type the peptide not an α-helical the of the N-terminal domain in a monomeric state is in an unordered on the it that was at the corresponding to the N-terminal domain of the wild-type protein the N-terminal domain from the RecA molecule (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). The of a local folding of the N-terminal domain to a of the self-association process. By the for an between the monomers and to the observed the dissociation constant for the interaction between RecA monomers was determined to be approximately 0.1 μm. be for of the self-association of RecA. important of the is that the N-terminal domain is involved in kinetic regulation of the self-association process. We suggested that the N-terminal of RecA an important role not only in protein-protein interactions but also in kinetic regulation of the self-association process (17Mikawa T. Masui R. Ogawa T. Ogawa H. Kuramitsu S. J. Mol. Biol. 1995; 250: 471-483Crossref PubMed Scopus (37) Google Scholar). treatment with a low concentration of urea in a of it is that an between oligomeric states of RecA is reduced by the unfolding of the N-terminal has been that the process of RecA is and that a of molecules is required to S.L. Zlotnick A. Stafford III, W.F. J. Mol. Biol. 1990; 216: 949-964Crossref PubMed Scopus (42) Google Scholar). are well ordered molecules in the between monomers (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). the absence or unfolding of the N-terminal which complementary in the in a decrease in the of bound to for interactions between RecA Although the in the self-association nucleoprotein filament have not been it has been that the oligomeric structure into oligomeric structures, prior to filament S.L. Zlotnick A. Stafford III, W.F. J. Mol. Biol. 1990; 216: 949-964Crossref PubMed Scopus (42) Google Scholar). these include the the folding of the N-terminal domain have to the oligomeric of RecA. has been that a peptide corresponding to the first 24 amino acid residues of RecA to ssDNA, which is with a folding from an unordered structure to an A. S.L. J. Mol. Biol. 1988; Scopus (25) Google Scholar). we showed that of an α-helical by the interaction not only with Δ33RecA but also with is or not the N-terminal is involved in binding to ssDNA, the structure of the RecA-DNA complex has not been The central and C-terminal in to the N-terminal be involved in protein-protein interactions and the structural to obtain information about these of the these are by of the of the residues and of the residues are in those The of the first on in urea denaturation of RecA was on the protein concentration the RecA was with m urea, the of the protein to was to even the N-terminal domain was the in the is to be with the protein-protein interaction the is to the folding state of the N-terminal The coli RecA protein and two residues (7Story R.M. Weber I. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar). is in the of the central and are on the of the between contiguous RecA molecules in the crystal structure and it that these residues to the spectrum in the The other three and residues are in the C-terminal The of the of these residues be is the a or conformational be to in the C-terminal We are to M. and Y. and for with the of