Membrane topology of the DrrB protein of the doxorubicin transporter of Streptomyces peucetius

Daunorubicin and doxorubicin, two commonly used anticancer agents, are produced by the soil bacterium Streptomyces peucetius. Self-resistance to these antibiotics in S. peucetius is conferred by the drrAB locus that codes for two proteins, DrrA and DrrB. DrrA is an ATP-binding protein. It belongs to the ABC family of transporters and shares sequence and functional similarities with P-glycoprotein of cancer cells. DrrB is an integral membrane protein that might function as a transporter for the efflux of daunorubicin and doxorubicin. Together, DrrA and DrrB are believed to form an ATP-driven pump for the efflux of these drugs. The drrAB locus has been cloned, and the two proteins have been expressed in a functional form in Escherichia coli. A topological analysis of the DrrB protein was performed using gene fusion methodology. Random and site-directed fusions of the drrB gene to lacZ, phoA, or gfp reporter genes were created. Based on the fusion data, a topological model of the DrrB protein is proposed in which the protein has eight membrane-spanning domains with both the N terminus and the C terminus in the cytoplasm.     

Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif

DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.     

The Q-loop of DrrA is involved in producing the closed conformation of the nucleotide binding domains and in transduction of conformational changes between DrrA and DrrB

DrrA and DrrB proteins form an ATP-dependent efflux pump for doxorubicin and daunorubicin in Streptomyces peucetius. DrrA, the catalytic subunit, forms a complex with the integral membrane protein DrrB. Previous studies have provided evidence for strong interaction between these two proteins, which was found to be critical for binding of ATP to DrrA and for stability of DrrB. Chemical cross-linking experiments carried out previously showed that in the resting state of the complex DrrA and DrrB are in contact with each other. Use of a cysteine-to-amine cross-linker then allowed identification of the N-terminal cytoplasmic tail of DrrB (residues 1-53) as the primary region of contact with DrrA. In this study, single-cysteine substitutions were introduced into different domains of DrrA in a strain already containing the S23C substitution in the N-terminal tail of DrrB. By using different arm-length disulfide cross-linkers, we found that a cysteine placed in the Q-loop region of DrrA traps DrrA in the dimeric state, thus indicating that in the closed conformation the Q-loops from opposing subunits are in the proximity of each other. Furthermore, the same region of DrrA was also found to interact with the N-terminus of DrrB, although the A-A interaction was much more prominent than the A-B interaction under these conditions. On the basis of additional data shown here, we propose that the interaction of the Q-loop with the N-terminal cytoplasmic tail of DrrB identifies an important step in the communication of conformational changes between DrrA and DrrB. The significance of these findings in the mechanism of the DrrAB complex is discussed, and a model based on analyses of different conformations of DrrA and DrrB is presented.     

The Extreme C Terminus of the ABC Protein DrrA Contains Unique Motifs Involved in Function and Assembly of the DrrAB Complex

Two novel regulatory motifs, LDEVFL and C-terminal regulatory Glu (E)-rich motif (CREEM), are identified in the extreme C terminus of the ABC protein DrrA, which is involved in direct interaction with the N-terminal cytoplasmic tail of the membrane protein DrrB and in homodimerization of DrrA. Disulfide cross-linking analysis showed that the CREEM and the region immediately upstream of CREEM participate directly in forming an interaction interface with the N terminus of DrrB. A series of mutations created in the LDEVFL and CREEM motifs drastically affected overall function of the DrrAB transporter. Mutations in the LDEVFL motif also significantly impaired interaction between the C terminus of DrrA and the N terminus of DrrB as well as the ability of DrrA and DrrB to co-purify, therefore suggesting that the LDEVFL motif regulates CREEM-mediated interaction between DrrA and DrrB and plays a key role in biogenesis of the DrrAB complex. Modeling analysis indicated that the LDEVFL motif is critical for conformational integrity of the C-terminal domain of DrrA and confirmed that the C terminus of DrrA forms an independent domain. This is the first report which describes the presence of an assembly domain in an ABC protein and uncovers a novel mechanism whereby the ABC component facilitates the assembly of the membrane component. Homology sequence comparisons showed the presence of the LDEVFL and CREEM motifs in close prokaryotic and eukaryotic homologs of DrrA, suggesting that these motifs may play a similar role in other homologous drug and lipid export systems.     

Translational Coupling Controls Expression and Function of the DrrAB Drug Efflux Pump

This study investigates the role of translational coupling in the expression and function of DrrA and DrrB proteins, which form an efflux pump for the export of anticancer drugs doxorubicin and daunorubicin in the producer organism Streptomyces peucetius. Interest in studying the role of translational coupling came from the initial observation that DrrA and DrrB proteins confer doxorubicin resistance only when they are expressed in cis. Because of the presence of overlapping stop and start codons in the intergenic region between drrA and drrB, it has been assumed that the translation of drrB is coupled to the translation of the upstream gene drrA even though direct evidence for coupling has been lacking. In this study, we show that the expression of drrB is indeed coupled to translation of drrA. We also show that the introduction of non-coding sequences between the stop codon of drrA and the start of drrB prevents formation of a functional complex, although both proteins are still produced at normal levels, thus suggesting that translational coupling also plays a crucial role in proper assembly. Interestingly, replacement of drrA with an unrelated gene was found to result in very high drrB expression, which becomes severely growth inhibitory. This indicates that an additional mechanism within drrA may optimize expression of drrB. Based on the observations reported here, it is proposed that the production and assembly of DrrA and DrrB are tightly linked. Furthermore, we propose that the key to assembly of the DrrAB complex lies in co-folding of the two proteins, which requires that the genes be maintained in cis in a translationally coupled manner.     

Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima.

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.     

Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius.

Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important antitumor drugs, were cloned from Streptomyces peucetius (the daunorubicin producer) and S. peucetius subsp. caesius (the doxorubicin producer) by use of the actI/tcmIa and actIII polyketide synthase gene probes. Restriction mapping and Southern analysis of the DNA cloned in a cosmid vector established that the DNA represented three nonoverlapping regions of the S. peucetius subsp. caesius genome. These three regions plus an additional one that hybridized to the same probes are present in the S. peucetius genome, as reported previously (K. J. Stutzman-Engwall and C. R. Hutchinson, Proc. Natl. Acad. Sci. USA 86:3135-3139, 1989). Functional analysis of representative clones from some of these regions in S. lividans, S. peucetius ATCC 29050, S. peucetius subsp. caesius ATCC 27952, and two of its blocked mutants (strains H6101 and H6125) showed that many of the antibiotic production genes reside in the region of DNA represented by the group IV clones. This conclusion is based on the production of epsilon-rhodomycinone, a key intermediate of the daunorubicin pathway, in certain S. lividans transformants and on the apparent complementation of mutations that block daunorubicin biosynthesis in strains H6101 and H6125. Some of the transformants of strains 29050, 27952, and H6125 exhibited substantial overproduction of epsilon-rhodomycinone and daunorubicin.     

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production.

The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.     

Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces

We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952, which produces clinically important anthracycline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. Among those, 15 code for functional genes, whereas the remaining four are pseudo genes. CYPs from S. peucetius are phylogenetically close to those of Streptomyces amermitilis. Four CYPs are associated with modular PKS of avermectin and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). Nine CYPs from S. peucetius are found in the cluster containing various regulatory genes including rar operon, conserved in S. coelicolor A3 (2) and Streptomyces griseus. Although two ferredoxins and four ferredoxin reductases have been identified so far, only one ferredoxin reductase was found in the cluster of CYP147F1 in S. peucetius. To date, 174 CYPs have been described from 45 Streptomyces species in all searchable databases. However, only 18 CYPs are clustered with ferredoxin. The comparative study of cytochrome P450s, ferredoxins, and ferredoxin reductases should be useful for the future development and manipulation of antibiotic biosynthetic pathways.     

Isolation of a chitinase overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Streptomyces peucetius, producer of the antitumor anthracycline antibiotic daunorubicin, was mutagenized, and mutants defective in daunorubicin biosynthesis were screened. One mutant (SPVI), which failed to produce daunorubicin, was found to overproduce an extracellular chitinase. Time course analyses of chitinase production and of the extracellular protein profile showed that the increase in activity is due to increased synthesis of the enzyme protein. The production of chitinase in SPVI was repressed by glucose as in the case of wild-type S. peucetius. PFGE analysis of VspI restriction fragments of S. peucetius and SPVI showed that there was no major alteration in the mutant genome. The hybridization pattern of S. peucetius and SPVI genomic DNA digested with various restriction enzymes was identical when probed with dnrUVJI genes of the S. peucetius daunorubicin cluster and chiA of Streptomyces lividans 66. The possible step affected in the daunorubicin biosynthetic pathway could be a polyketide synthase, since aklanonic acid, the earliest detectable intermediate in the daunorubicin pathway, was not synthesized in SPVI.     

Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius.

Two DNA segments, dnrR1 and dnrR2, from the Streptomyces peucetius ATCC 29050 genome were identified by their ability to stimulate secondary metabolite production and resistance. When introduced into the wild-type ATCC 29050 strain, the 2.0-kb dnrR1 segment caused a 10-fold overproduction of epsilon-rhodomycinone, a key intermediate of daunorubicin biosynthesis, whereas the 1.9-kb dnrR2 segment increased production of both epsilon-rhodomycinone and daunorubicin 10- and 2-fold, respectively. In addition, the dnrR2 segment restored high-level daunorubicin resistance to strain H6101, a daunorubicin-sensitive mutant of S. peucetius subsp. caesius ATCC 27952. Analysis of the sequence of the dnrR1 fragment revealed the presence of two closely situated open reading frames, dnrI and dnrJ, whose deduced products exhibit high similarity to the products of several other Streptomyces genes that have been implicated in the regulation of secondary metabolism. Insertional inactivation of dnrI in the ATCC 29050 strain with the Tn5 kanamycin resistance gene abolished epsilon-rhodomycinone and daunorubicin production and markedly decreased resistance to daunorubicin. Sequence comparison between the products of dnrIJ and the products of the Streptomyces coelicolor actII-orf4, afsR, and redD-orf1 genes and of the Streptomyces griseus strS, the Saccharopolyspora erythraea eryC1, and the Bacillus stearothermophilus degT genes reveals two families of putative regulatory genes. The members of the DegT, DnrJ, EryC1, and StrS family exhibit some of the features characteristic of the protein kinase (sensor) component of two-component regulatory systems from other bacteria (even though none of the sequences of these four proteins show a significant overall or regional similarity to such protein kinases) and have a consensus helix-turn-helix motif typical of DNA binding proteins. A helix-turn-helix motif is also present in two of the proteins of the other family, AfsR and RedD-Orf1. Both sets of Streptomyces proteins are likely to be trans-acting factors involved in regulating secondary metabolism.     

The DrrC protein of Streptomyces peucetius, a UvrA-like protein, is a DNA-binding protein whose gene is induced by daunorubicin

DrrC, a daunorubicin resistance protein with a strong sequence similarity to the UvrA protein involved in excision repair of DNA, is induced by daunorubicin in Streptomyces peucetius and behaves like an ATP-dependent, DNA binding protein in vitro. The refolded protein obtained from expression of the drrC gene in Escherichia coli was used to conduct gel retardation assays. DrrC bound a DNA segment containing the promoter region of a daunorubicin production gene only in the presence of ATP and daunorubicin. This result suggests that DrrC is a novel type of drug self-resistance protein with DNA binding properties like those of UvrA. Western blotting analysis with a polyclonal antiserum generated against His-tagged DrrC showed that the appearance of DrrC in S. peucetius is coincident with the onset of daunorubicin production and that the drrC gene is induced by daunorubicin. These data also showed that the DnrN and DnrI regulatory proteins are required for drrC expression. The level of DrrA, another daunorubicin resistance protein that resembles ATP-dependent bacterial antiporters, was regulated in the same way as that of DrrC.     

Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius

The DnrI protein, essential for the biosynthesis of daunorubicin in Streptomyces peucetius , was purified almost to homogeneity from dnrI expression strains of Escherichia coli and S. peucetius through several steps of chromatography. The proteins purified from both organisms had identical chromatographic and electrophoretic behaviour. Purified His-tagged or native DnrI was used to conduct DNA-binding assays by gel mobility-shift analysis, and the results showed no significant difference in the DNA-binding activity of native or His-tagged proteins. DnrI binds specifically to DNA segments containing the intergenic regions separating the putative dnrG–dpsABCD and dpsEF operons, and the dnrC gene and dnrDKPSQ operon. DNase I footprinting assays indicated that the DNA-binding sites for DnrI extended from upstream of the −10 to −35 regions of the dnrG or dpsE promoters to include about 65 bp of the dnrG – dpsE intergenic region and about 80 bp of the dnrC – dnrD intergenic region. Both binding sites contain imperfect inverted repeat sequences of 6–10 bp with a 5'-TCGAG-3' consensus sequence that was present in 4 out of 10 other promoter regions in the cluster of daunorubicin biosynthesis genes.     

High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum‐derived vesicles to Legionella‐containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4‐phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P‐binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.     

The Streptomyces peucetius dpsY and dnrX Genes Govern Early and Late Steps of Daunorubicin and Doxorubicin Biosynthesis

The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.     

Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia coli.

Sequence analysis of a portion of the Streptomyces peucetius daunorubicin biosynthetic gene cluster revealed a complete open reading frame (dnrK) that showed DNA and protein sequence homology to several O-methyltransferases. Expression of dnrK in Streptomyces lividans and Escherichia coli was done to show that this gene codes for carminomycin 4-O-methyltransferase. The deduced carminomycin 4-O-methyltransferase protein shows a conserved nucleotide binding site for its S-adenosyl-L-methionine cofactor.     

天蓝淡红链霉菌SIPI-1482的dauW基因的克隆及在大肠杆菌中的表达

克隆得到了柔红霉素产生菌天蓝淡红链霉菌(Streptomyces coeruleorubidus) SIPI-1482位于dnrX下游的新基因dauW,其位于基因组上dnrX和drrB之间,GenBank中Blast发现它与dnrW有较高的同源性,将其命名为dauW,并提交GenBank获得登录号EF523565,根据保守域推测dauW所编码的蛋白属于FAD依赖的氧化还原酶类。将dauW分别克隆至表达载体pET-28a(+)、pET-32a(+),在宿主菌BL21(DE3)中经IPTG诱导后,实现了在大肠杆菌中的表达。初步实验表明dauW在BL21(DE3)中的表达能增加宿主对柔红霉素的抗性,可能与天蓝淡红链霉菌对柔红霉素的自身抗性有关。     

Nucleotide-induced conformational changes in the human multidrug resistance protein MRP1 are related to the capacity of chemotherapeutic drugs to accumulate or not in resistant cells

Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface. Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug-resistant cells. On the contrary, three compounds (4'-deoxy-4'-iodo-doxorubicin, 4-demethoxy-daunorubicin and 3'-(3-methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/MRP1 cells. Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1-mediated drug transport. MRP1 was reconstituted in lipid vesicles as previously described [Manciu, L., Chang, X.B., Riordan, J.R. and Ruysschaert, J.-M. (2000) Biochemistry 39, 13026-13033]. The reconstituted protein was shown to conserve its ATPase and drug transport activity. Acrylamide quenching of Trp fluorescence was used to monitor drug-dependent conformational changes. Binding of drugs (4-demethoxy-daunorubicin and 3'-(3-methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process.     

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

GDP‐bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub‐cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP‐to‐GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA‐1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure‐based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.