Biochemical Coupling between the DrrA and DrrB Proteins of the Doxorubicin Efflux Pump of Streptomyces peucetius

Abstract

The drrAB operon ofStreptomyces peucetius encodes for resistance to the antibiotics doxorubicin and daunorubicin. Subcloning of thedrrAB genes in Escherichia coli has previously been shown to result in expression of DrrA and DrrB proteins and resistance to doxorubicin in a sensitive strain of E. coli. DrrA, a peripheral membrane protein, binds ATP in a UV-catalyzed reaction in a doxorubicin-dependent manner; DrrB, a hydrophobic protein, is localized to the inner membrane of E. coli (Kaur, P. (1997) J. Bacteriol. 179, 569–575). The present study provides evidence that DrrB, the membrane component of the complex, is stably maintained in the cell only if DrrA is present. Furthermore, it was found that the catalytic component DrrA is in an active conformation only when it is in a complex with DrrB. In a subclone containing the drrB gene by itself, no DrrB protein could be detected, although a translational fusion of the first 15 amino acids of DrrB to β-galactosidase indicated that DrrB is translated in the absence of DrrA. Upon co-transformation with a plasmid containing the drrA gene in trans, DrrB could again be detected in these cells. UV cross-linking studies with [α-32P]ATP showed that only the membrane-bound form of DrrA in cells containing both DrrA and DrrB was in a conformation competent to bind ATP. Chemical cross-linking studies also provided direct evidence for interaction between the two proteins. Based on these analyses, a model for interaction between DrrA and DrrB proteins is presented. The drrAB operon ofStreptomyces peucetius encodes for resistance to the antibiotics doxorubicin and daunorubicin. Subcloning of thedrrAB genes in Escherichia coli has previously been shown to result in expression of DrrA and DrrB proteins and resistance to doxorubicin in a sensitive strain of E. coli. DrrA, a peripheral membrane protein, binds ATP in a UV-catalyzed reaction in a doxorubicin-dependent manner; DrrB, a hydrophobic protein, is localized to the inner membrane of E. coli (Kaur, P. (1997) J. Bacteriol. 179, 569–575). The present study provides evidence that DrrB, the membrane component of the complex, is stably maintained in the cell only if DrrA is present. Furthermore, it was found that the catalytic component DrrA is in an active conformation only when it is in a complex with DrrB. In a subclone containing the drrB gene by itself, no DrrB protein could be detected, although a translational fusion of the first 15 amino acids of DrrB to β-galactosidase indicated that DrrB is translated in the absence of DrrA. Upon co-transformation with a plasmid containing the drrA gene in trans, DrrB could again be detected in these cells. UV cross-linking studies with [α-32P]ATP showed that only the membrane-bound form of DrrA in cells containing both DrrA and DrrB was in a conformation competent to bind ATP. Chemical cross-linking studies also provided direct evidence for interaction between the two proteins. Based on these analyses, a model for interaction between DrrA and DrrB proteins is presented. Doxorubicin and daunorubicin, two antibiotics produced byStreptomyces peucetius, are widely used in chemotherapy.S. peucetius is itself resistant to the inhibitory action of these drugs. Resistance in S. peucetius is encoded by thedrr operon that contains two open reading frames,drrA and drrB (1Guilfoile P.G. Hutchinson R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8553-8557Crossref PubMed Scopus (162) Google Scholar). DrrA and DrrB proteins have been hypothesized to form an efflux pump that confers resistance by transport of the drugs out of the cell (1Guilfoile P.G. Hutchinson R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8553-8557Crossref PubMed Scopus (162) Google Scholar, 2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). DrrA protein consists of one nucleotide binding domain with homology to the ABC family of proteins to which P-glycoprotein (Pgp 1The abbreviations used are: Pgp, P-glycoprotein; PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl-1-thio-β-d-galactopyranoside; DSP, dithiobis(succinimidyl propionate); DTSSP, dithiobis(sulfosuccinimidyl propionate). ; Ref. 3Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar) belongs. The product of the drrB gene is a membrane protein (1Guilfoile P.G. Hutchinson R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8553-8557Crossref PubMed Scopus (162) Google Scholar, 2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar) with homology to membrane components of the bacterial ABC transporters (4Reizer J. Reizer A. Saier M.H. Protein Sci. 1992; 1: 1326-1332Crossref PubMed Scopus (88) Google Scholar). Pgp is expressed in human cancer cell lines, where it confers resistance to a variety of structurally unrelated chemotherapeutic drugs, including doxorubicin and daunorubicin (3Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar). This phenomenon is referred to as multidrug resistance. Pgp contains a drug-stimulated ATPase activity (5Senior A.E. Al-Shawi M.K. Urbatsch I.L. J. Bioenerg. Biomembr. 1995; 27: 31-36Crossref PubMed Scopus (71) Google Scholar); however, its mode of action and the mechanism by which it can transport structurally unrelated substrates is not understood. Since DrrA and Pgp are homologous proteins and both confer resistance to doxorubicin and daunorubicin, an understanding of the structure and function of Drr pump is expected to contribute to a better understanding of the function and evolution of Pgp. The drr operon has previously been subcloned in anEscherichia coli expression vector, and both proteins have been identified using antisera raised in rabbits (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). Expression of the proteins in an acr strain of E. coli resulted in doxorubicin resistance in these cells, indicating that the proteins expressed in E. coli retain proper conformation and function. Isolation of active alkaline phosphatase fusions in DrrB showed that DrrB is an integral membrane protein (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). DrrA, a peripheral membrane protein was shown to bind ATP or GTP in the presence of Mg2+. The nucleotide binding to DrrA was found to be doxorubicin-dependent (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar), indicating that the activity of DrrA is regulated by the substrate of the pump. Most ABC transporters, including Pgp, consist of two nucleotide binding domains and two membrane-spanning domains of six α-helices each. In Pgp, the four domains form one large protein of about 170 kDa (3Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3562) Google Scholar). The Drr system, instead, consists of two subunits, DrrA and DrrB, that carry individual domains. Together, a monomer of DrrA and a monomer of DrrB form a molecule about half the size of Pgp; however, the stoichiometry of the DrrA and DrrB proteins in the Drr pump is not known. The present study focuses on an interaction between DrrA and DrrB; the data indicate a strong dependence of the proteins on each other for their expression and function such that the two protein subunits behave as domains of a larger protein. The DrrB protein was detected in the cell membrane of these cells only if there was simultaneous expression of DrrA. In addition, DrrA was in an active conformation to bind and/or hydrolyze ATP only if it was in a complex with DrrB in the membrane. Chemical cross-linking studies also suggest a direct interaction between DrrA and DrrB with the suggested stoichiometry of the complex being DrrA2B2. These results bear important implications for the evolution of multidomain proteins, including Pgp, and for understanding the interaction between their domains. The E. colistrains and plasmids used in this study are described in Table I.Table IStrains and plasmidsStrain or plasmidGenotype or descriptionReference or sourceStrain TG1K12 Δ(lac-pro), supE, thi, hsd D5/F′ tra36, pro A+B+, lacIq, lacZΔM157Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar CC118F− araD139Δ(ara-leu)7697 ΔlacX74 phoAΔ20 galE15 galK16 thi rpsE rpoB argE(Am) recA111Manoil C. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8129-8133Crossref PubMed Scopus (659) Google ScholarPlasmid pUC18arsAarsA, Ampr12Kaur P. Rosen B.P. J. Bacteriol. 1993; 175: 351-357Crossref PubMed Google Scholar pMC1069Vector, Ampr6Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar pDx101drrAB in pSU2718arsA, Cmr2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar pDx102drrA in pSU2718arsA, Cmr2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar pDx103drrB in pSU2718arsA, Cmr2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar pDX108drrA in pUC18arsA, AmprThis study pDX109drrA and 45 ntant, nucleotide(s). from the 5′-end ofdrrB fused to 24th nt of lacZ in pMC1069This study pDX11045 nt from the 5′-end of drrB fused to 24 nt of lacZ in pMC1069This studya nt, nucleotide(s). Open table in a new tab The cells were grown in LB medium (6Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar) at 37 °C. Chloramphenicol was added to 20 μg/ml, and ampicillin was added to 100 μg/ml. The conditions for plasmid isolation, DNA endonuclease restriction analysis, ligation, and sequencing have been described elsewhere (7Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The 1.0-kilobase pairNdeI–HindIII fragment from pDX102 was subcloned behind the lac promoter in pUC18 vector by substitution of the arsA gene in pUC18arsA. This clone has been designated pDX108. The drrA gene in this construct utilizes the translational start signals of the arsA gene. A translational fusion of the first 15 amino acids of DrrB to the 8th amino acid of β-galactosidase was created. Using pDX101 or pDX103 as the template (Fig. 1 and Table I), the region contained between the lac promoter and the 45th nucleotide of the drrB gene was polymerase chain reaction-amplified. Polymerase chain reaction primers had the following nucleotide sequence, and each primer contained the following HindIII restriction site: I, 5′-GCCGCCAAGCTTCCTCTCCCCGCGCGTTGG-3′; II, 5′-GCCGCCAAGCTTCGCTCACAGGGGTCGTGG-3′. The amplified product was ligated to the translational fusion vector pMC1069 that had been digested withHindIII. Amplification and subcloning of the fragment from pDX101 and pDX103 resulted in plasmids pDX109 and pDX110, respectively. pDX109 contains plac and the ribosome binding site ofarsA, followed by the drrA gene translationally coupled to drrB-lacZ fusion, as is seen in the original operon. The drrA gene, however, is missing from pDX110. CC118 cells containing pDX109 or pDX110 were grown to mid-log phase and induced with 1 mm IPTG for 3 h. The cells were harvested and lysed by a single passage through a French pressure cell at 20,000 p.s.i. The lysed cells were centrifuged at 10,000 × g for 15 min to remove unbroken cells. 100 μg of protein was then added to a tube containing 0.5 ml of Z buffer containing 60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm of and the reaction was for The reaction was with 0.5 ml of 1 were as described (6Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). E. coli cells containing the indicated plasmids were grown to mid-log phase and induced with mm was for an at 37 °C. The cells were and lysed by a single passage through a French pressure cell at 20,000 p.s.i. The cell were centrifuged at 10,000 × g for 10 min to remove the unbroken cells. The were used to the as described (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). The were in buffer by at for 10 The proteins were by on polyacrylamide followed by as described (1Guilfoile P.G. Hutchinson R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8553-8557Crossref PubMed Scopus (162) Google Scholar). of protein on the was out by using the Protein and DNA μg of membrane protein was with the indicated of in 1 ml of buffer containing mm The were on for The were by at × g for 1 and in mm The were by at for 10 The proteins were by on polyacrylamide followed by as described of [α-32P]ATP to the DrrA protein was out by the described (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar, PubMed Scopus Google Scholar). was in the and membrane from cells DrrA or both DrrA and DrrB proteins. The were used in at the indicated protein Doxorubicin was added to the where The were by followed by The reaction contained cell membrane protein in a buffer containing 20 mm and mm or was added at the indicated and the were at for The were by the of a of 1 to a of followed by an at for 15 buffer PubMed Scopus Google Scholar) the was and the were by using a polyacrylamide followed by with or (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). carry out of the a from the gel was and in the buffer containing for 20 min at The proteins were by the on of an polyacrylamide The were with to DrrB, followed by with to DrrA. Expression of the DrrA or DrrB DrrA and DrrB proteins have previously been shown to be expressed in E. coli from the lac promoter in pDX101 In the present expression of DrrA or DrrB was in pDX102 and which the drrA and drrB gene, respectively. A of these is in 1 A. The genes in or the lac promoter and the translational start signals of the arsA gene. pDX101 also contains a ribosome binding site for which is contained the of In addition, the region between drrA and drrB as in 1 translational such that the of drrA with the start is in in pDX103 its and it utilizes the of E. coli cells containing the indicated plasmids were induced with IPTG, and were 3 of The cells were and membrane The were by using or (Fig. 1 and the of the were by using suggested that DrrA is expressed both in and however, the of expression in pDX102 (Fig. 1 1 and was in pDX101 (Fig. 1 suggested that the of DrrA in pDX102 was about that of in in both in the presence or the absence of DrrB, DrrA was A of DrrA that with (Fig. 1 is unrelated to DrrA, this is also in the that not DrrA or DrrB (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). by using suggested that DrrB is present only in cells (Fig. 1 3 and if DrrB could be detected in the membrane from cells containing pDX103 (Fig. 1 1 and if the expression of DrrB in pDX101 is the result of translational with DrrA and the absence of DrrB in pDX103 is a result of the absence a translational fusion of the first 15 amino acids of DrrB with β-galactosidase was created. Using pDX101 or pDX103 as a the region at the promoter plac and with the 45th nucleotide of drrB was amplified by polymerase chain and the amplified fragment was fused to the of from this (Fig. one containing gene in and the other were for β-galactosidase The data in indicate that drrB in pDX110 can be although the β-galactosidase activity in where drrA is present in was about as with pDX110. Since DrrB is translated in the absence of coupled with DrrA, not detected in these cells by (Fig. to if of DrrB in the cell on simultaneous expression of DrrA. E. coli cells containing pDX103 were with a plasmid containing the drrA gene. contains gene in pUC18 as in the translational start signals of arsA and of the drrA and drrB genes with IPTG, cell were for the presence of DrrA and DrrB proteins. in 3 that the expression of DrrB from pDX103 is in cells containing DrrA in The of DrrB protein detected was the the drrA gene was present in or and indicating that the presence of the DrrA protein is for of DrrB in the is also seen in the of DrrA in cells was (Fig. as with cells (Fig. expression of DrrA in results in the of DrrA protein as in pDX101 (Fig. This be to the the of plasmid pUC18 in with the plasmid in The at kDa is the protein with the and it is present in of the The of the of DrrA or DrrB to the cell membrane was by with (Fig. DrrA was with in both and (Fig. and it could be by in both 3 and DrrB was not at by (Fig. the that DrrB is an integral membrane protein, DrrA is with the membrane in both or cells. has previously been shown that DrrA can form a with ATP or GTP in a doxorubicin-dependent in cell containing both DrrA and DrrB (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). if DrrB ATP binding to DrrA, ATP binding was in the and membrane from E. coli cells containing pDX101 or [α-32P]ATP binding in a UV-catalyzed cross-linking showed that DrrA to ATP only in from cells containing both DrrA and DrrB (Fig. 3 and ATP cross-linking was seen to DrrA in containing DrrA (Fig. 1 and of a gel using to DrrA or DrrB showed that DrrA was present in (Fig. DrrB was present only in 3 and (Fig. it was the of DrrA in cells containing both DrrA and DrrB can be to ATP. in and that the of DrrA in cells was also not in a competent to to ATP. the membrane form of DrrA in cells was competent (Fig. and showed that DrrA was present in both the and the membrane (Fig. DrrB was present only in the membrane (Fig. other in the that with with DrrA, were also seen to form a with ATP (Fig. and of these are unrelated to DrrA and bind ATP of the presence or absence of DrrB. Chemical cross-linking was out in the cell from cells. of were the and the and can be in the presence of the of the from to were used to interaction between DrrA and DrrB. with both the were (Fig. Upon of the gel conditions and using two were identified (Fig. DrrB, a protein, at kDa as has also been (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). The of one was about 60 kDa with of about kDa was also with a could be detected at of both as as as (Fig. and the at about 100 3 and with a kDa was also with a This was at of the and it the at 1 and about mm of The were not when from cells containing the vector, were with the not Upon of a gel with the DrrA was seen to at about kDa as expected (Fig. The seen in A with could be seen to with the (Fig. with an indicating that this is a complex containing both DrrA and DrrB. The reaction of the with the was in the first gel shown in Since and are of the and conditions was out to the presence of DrrA and DrrB in these (Fig. to the in in was in the presence of the as described was on of gel and to conditions on a the proteins on the Upon the the the proteins on the In A and with The gel in A is the of the the in has been and in are the in A and that have been with are seen on the in A. These to the DrrB and the described in These are again with and the with an to the DrrA. Upon of the proteins, followed by and with DrrB on the (Fig. of the was to a that with the as DrrB. if the of these also resulted in DrrA, the in was with The data shown in that the DrrA on the however, of each also DrrA in to DrrB as seen in of the from the gel in was and the were by with The was on of a gel and to followed by with and A and and with The of the is with and on the in A In the present expression and function of the DrrA and DrrB proteins was found to be on each in a strong In cells containing the plasmid which contains drrA and drrB genes in both DrrA and DrrB proteins were Upon subcloning of gene in DrrA was detected at in indicating that DrrB the expression or of DrrA. of DrrA on DrrB expression or however, was Subcloning of gene in pDX103 showed the absence of DrrB protein (Fig. Since and the the of expression of drrB to be at the of or DrrA in the original operon are translationally coupled that the of drrA with the start ofdrrB (Fig. if is for expression of a translational fusion containing first 15 amino acids of DrrB fused to the of β-galactosidase was created. of the fusion indicated that DrrB is translated in the absence of DrrA in pDX110 although at in pDX109 that contains DrrA in (Fig. translational not to be for of DrrB, although it to have in of of DrrB in showed that the simultaneous expression of DrrA, from a gene in in trans, was for of DrrB in the drrA and drrB genes were the cell on DrrB protein, translated from was again detected at the as in pDX101 (Fig. that the is the of translational in pDX101 and pDX109 of DrrB, it is important to the of the This is however, it is that drrB is translated in the protein is not detected in these cells by gene is these cells, DrrB is again detected at the as in there to be a mechanism by which DrrB is in DrrA is not present. studies have that of DrrB by itself as a translational fusion to results in of cell a of its (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). This could be to a membrane from a open by DrrB in the absence of DrrA, which the for a mechanism to DrrB, its Experiments are to the mechanism by which DrrA DrrB in the Since found strong evidence for a interaction between DrrA and DrrB, cross-linking studies were out to the interaction and to the stoichiometry of the complex in the membrane. out with both a and a showed the presence of two one of 60 kDa and of were shown by analysis, to consist of both DrrA and DrrB. DrrA as a protein, and DrrB, a protein, at the to a complex of one of DrrA and one of DrrB, the consists of a complex of two subunits of DrrA and two subunits of DrrB. A with a a containing DrrA, DrrB, and other membrane proteins, was also the of this was seen to at of the it the at 1 the of the a strong interaction between DrrA and DrrB. Based on the it can also be that the complex consists of two subunits of DrrA and two of DrrB. The to to or that in the Drr complex, DrrA only DrrB and not of DrrA and that the complex has the of where the 1 or indicate 1 or the This the pump to Pgp in of the of domains and the both containing two ABC and two membrane-spanning domains. The that the interaction is between and or between and the the and also be expected these were not detected, a between and results in the of or to the of and or and between subunits of transport has been J. J. Bacteriol. 1997; 179: PubMed Google Scholar); however, such a of expression and function as seen in the has not been has been that the transport as of subunits that are encoded by genes an the transporters as single large proteins that domains. a consists of two expressed however, the subunits are coupled and to behave the domains of a single protein. In to DrrA the or membrane of DrrB, the function of DrrA itself was found to be its interaction with DrrB. DrrA has previously been shown to be an protein (2Kaur P. J. Bacteriol. 1997; 179: 569-575Crossref PubMed Google Scholar). In the present have shown that no ATP cross-linking to DrrA was seen in the or membrane-bound form of DrrA in cells containing only DrrA. ATP cross-linking to DrrA was seen only in the membrane-bound of DrrA in cells (Fig. These data suggest that it is the interaction between DrrA and DrrB in the membrane that a in the ATP binding site of DrrA. although DrrA is with the membrane in the absence of DrrB (Fig. it is not in an active the of DrrB to be stably maintained in the cell and/or the membrane on DrrA, and the of DrrA to bind and/or hydrolyze ATP on the presence of DrrB in the membrane. In this the complex is a ABC where two components each other in a Based on the data in this that DrrA is an protein that through on the presence or absence of DrrB and the doxorubicin (Fig. The DrrB protein is shown as of α-helices on of gene fusions with alkaline phosphatase and and J. In its DrrA is in conformation I, which is in of its to with ATP. with DrrB results in of DrrB in the cell and this interaction a in the active site of DrrA, which is shown as conformation conformation of DrrA, as conformation can be conformation the of is in this conformation that DrrA is to form the with ATP and A better understanding of DrrA and DrrB to a better understanding of domains Pgp and each other and be between the catalytic ABC domain and the membrane-spanning domain of Pgp. This has implications for understanding the evolution of multidomain proteins found in cells. are to P. C. for of the