The Extreme C Terminus of the ABC Protein DrrA Contains Unique Motifs Involved in Function and Assembly of the DrrAB Complex
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
09/2010; 285(49):38324-36. DOI: 10.1074/jbc.M110.131540
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.
Available from: Guoxing Fu
- "A comparison of the different crystal structures interestingly shows a high structural conservation in the region of the GATE domain. Specifically, we found that this region contains two b-sheets sandwiched by an a-helix or a loop on either side (Fig. 1C), which is also seen in the homology model of DrrA  (Fig. 1C.1). The highly conserved residue G215 (or the corresponding glycine) is present in the turn region between the two b-sheets in each structure which has been termed the 'Gly-loop' (G-loop) in this article. "
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ABSTRACT: A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homolog MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis.
Copyright © 2015. Published by Elsevier Inc.
Available from: Rong-Tzong Tsai
- "These ABC transporter domains can be located in a single polypeptide (such as Arabidopsis Comatose and cystic fibrosis transmembrane conductance regulator [CFTR]), in two polypeptides (such as human ALDP, the human transporters associated with antigen processing TAP1 and TAP2, and the yeast peroxisomal transporters Pxa1p and Pxa2p), or in four polypeptides (such as the Escherichia coli maltose transporter) –. In addition to the common ABC transporters TMD and NBD, some possess an extra sequence or accessory domain to regulate ATPase activity, channel opening, and interactions between NBD and NBD or NBD and TMD, as in the case of SUR2x/Kir6.2, maltose transporter MalFGK2, molybdate/tungstate transporter ModBC, human CFTR, and DrrA –. "
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ABSTRACT: The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette [ABC] transporter). This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p.
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- "However, the CREEM motif was absent from OtrC-ORF1 and Msr-ORF1. The LDEVFL motif is known to be involved in the doxorubicin-stimulated nucleotide-binding function and also exists in the MDR protein
. Therefore, it was speculated that the ATPase activity of OtrC-ORF1 is stimulated by doxorubicin and other drugs. "
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The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production.
In order to investigate OtrC’s function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively).
The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.
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