CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA

CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAPTER 7 – x RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA

135 CHAPTER X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHRISTOPHER B ROTH AND GEOFFREY A CHANG INTRODUCTION: THE MULTIDRUG RESISTANCE (MDR) ABC TRANSPORTERS ABC exporters transport a diverse array of substrates, including peptides, toxins, lipids and hydrophobic drug molecules, from the cytoplasmic side of the cell membrane to either the outer membrane leaflet or the outside of the cell Overexpression of a subset of these exporters is the most frequent cause of resistance to cytotoxic agents including antibiotics and anticancer drugs These ABC transporters have been likened to ‘hydrophobic vacuum cleaners’ because of their ability to remove drugs from the inner membrane leaflet (see Chapter 12) Many of these transporters are believed to be ‘flippases’, transporting or ‘flipping’ drugs and/or lipids from the inner to the outer membrane leaflet Some of the best-studied MDR-ABC transporters are the human P-glycoprotein (Pgp) and other related drug transporters Human Pgp is located in the plasma membrane of numerous cell types and transports a remarkably broad array of hydrophobic compounds (Ambudkar et al., 1999) Overexpression of human Pgp in the plasma membrane can confer resistance to chemotherapeutic drugs by intercepting them in the membrane and pumping them to the extracellular medium or the outer membrane leaflet Human Pgp is a 170 kDa polypeptide that has the four domains of a typical ABC transporter The two transmembrane domains each consist of six predicted membrane-spanning ␣-helices separated by hydrophilic loops These transmembrane domains (TMDs) are thought to recognize substrates and form a pathway by which hydrophobic compounds are transported across the cell membrane The nucleotide-binding domains (NBDs), are located at the cytoplasmic face of the membrane and couple ATP hydrolysis to substrate transport Recent advances in microbial genome sequencing projects have revealed prokaryotic ABC exporters that are similar in protein sequence to human Pgp Protein sequence alignment using the TMDs of selected ABC exporters using the program ClustalW reveals a distinct phylogenetic grouping (Figure 7.1) One of these, LmrA, from the bacterium Lactococcus lactis is a close functional homologue of human Pgp (van Veen et al., 1998) Whereas human Pgp consists of four fused domains, LmrA is organized as a homodimer of two polypeptides (van Veen et al., 2001) Hence, LmrA and most bacterial mdr-ABC transporters are called half *Parts of this chapter are reprinted with permission from the American Association for the Advancement of Science Please see Acknowledgments section for details ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 136 ABC PROTEINS: FROM BACTERIA TO MAN HLYB ECOLI MSBA VIBCH MSBA ECOLI MSBA HAEIN MSBA PASMU LMRA LACLA YVCC BACSU MDR1 HUMAN MDR3 HUMAN MDR2 MOUSE TAP1 HUMAN TAP2 HUMAN CYDD BACSU CFTR HUMAN CCMB ECOLI 0.1 Figure 7.1 Phylogenetic tree of the transmembrane domain portion of various ABC efflux pump genes (ClustalW with the PHYLIP output option) The GENBANK notations are used for gene and species designation transporters with each polypeptide containing a TMD and an NBD LmrA and the human Pgp extrude a remarkably similar spectrum of amphiphilic cationic compounds By protein sequence comparison using scores derived from the BLAST algorithm, the lipid flippase MsbA from Escherichia coli (Eco-MsbA) is one of the closest bacterial ABC transporters to human Pgp (Figure 7.1) The complete Eco-MsbA transporter is predicted to be a functional homodimer with a total molecular mass of 129.2 kDa MsbA transports lipid A, a major component of the bacterial outer cell membrane, and is essential for cell viability (Karow and Georgopoulos, 1993) Loss of MsbA expression in the cell membrane, or a disruption of transport by mutation, results in a lethal accumulation of lipid A in the cytoplasmic leaflet (Doerrler et al., 2001; Zhou et al., 1998) Numerous bacterial homologues of MsbA have been reported in more than 30 divergent prokaryotic species (McDonald et al., 1997) STRUCTURAL STUDIES OF MDR-ABC TRANSPORTERS Although a significant effort has been focused on understanding the biochemical mechanism of substrate translocation of ABC transporters, the evidence gathered to date has lacked a structural foundation on which to build a complete functional model Some of the key unresolved issues include the structural basis of coupling ATP hydrolysis to the movement of substrates and the achievement of specificity by the TMD One of the early pioneering attempts to generate a three-dimensional (3-D) model of an intact MDR-ABC transporter used cryo-electron microscopy to study both single particles and 2-D crystalline arrays of human Pgp (Rosenberg et al., 1997) In this groundbreaking work, a 25 Å EM structure revealed a significant chamber with no evidence of close contact between the NBDs Cryo-EM studies of Pgp trapped in distinct catalytic states revealed dramatic rearrangements of the TMD during the transport cycle Although the precise boundaries of the transmembrane and NBD elements cannot be unambiguously determined, there is a substantial opening in the plane of the cell membrane that was clearly resolved and appears to form a chamber that could accept substrates directly from the lipid bilayer The existence of a chamber within the bilayer is consistent with biochemical data supporting a ‘flippase’ model for human Pgp, and potentially other members of the MDR-ABC transporter family (Higgins and Gottesman, 1992) The recent X-ray structure of the lipid flippase MsbA from E coli at 4.5 Å in resolution establishes the overall structural architecture of an ABC transporter and suggests a model for the structural basis of the flipping mechanism that moves hydrophobic substrates from the inner to the outer membrane leaflet of the cell membrane (Chang and Roth, 2001) The close protein sequence homology to other MDRABC exporters strongly suggests a common mechanism that is general for this family As Eco-MsbA is the first member of the ABC transporter family to be elucidated by X-ray crystallography, we will overview some of the techniques and methods used for membrane protein structure determination X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA PROTEIN EXPRESSION PROTEIN PURIFICATION Membrane protein X-ray structure determination of transporters and ion channels presents new challenges in several areas including: the relatively lower natural abundance of these molecules in the cell membrane; difficulties in protein purification due to the presence of detergent; and disorder caused by inherent movements of the transmembrane ␣-helices Because of these difficulties, we adopted a general strategy of rapidly exploring crystallization space by cloning, overexpressing, and purifying more than 20 full-length bacterial ABC transporters from 12 bacterial species Our anticipation was that one or more of these naturally occurring proteins would be optimal for protein expression, purification and crystal formation Plasmids expressing full-length MsbA were generated by excision of the target genes amplified by polymerase chain reaction (PCR) The PCR products were cloned into Novagen pET 19b expression vector, which contains a leader sequence containing an amino-terminal decahistidine tag The ligated DNA product vector was then transformed into the XL-1 (Strategene) E coli strain Testing of protein expression was performed by growing a single clone overnight in Terrific Broth (TB; 12 g tryptone, 24 g yeast) containing carbenicillin (50 ␮g mlϪ1) and then transferring ml of this culture to TB for trial protein expression Cultures were induced for expression during mid-log phase (OD600 Ϸ 0.6–0.8) using isopropyl-␤-D-thiogalactoside (IPTG) Large-scale expression of MsbA was accomplished by batch fermentation in an 80-liter BioPilot (New Brunswick Scientific) A small overnight culture from a single bacterial clone was grown to OD600 Ϸ0.6–0.8 before transferring into the fermentor containing TB ϩ carbenicillin (100 ␮g/␮lϪ1) and 3% glycerol as a supplemental carbon source Oxygen saturation was maintained above 40% during fermentation by adjusting the agitation and air flow Protein expression was induced for hours with mM IPTG when the cell density approached an OD600 Ϸ7–8 The entire fermented culture was harvested in under an hour by centrifugation (6000 ϫ g) Cell pellets (0.6–1.2 kg) were flash frozen in liquid nitrogen and then stored at Ϫ80°C to facilitate lysing and subsequent purification The general purification strategy involved the (1) preparation of cell membranes, (2) solubilization in one or more detergents, and (3) protein purification by metal affinity, ion exchange, and size exclusion chromatography In the case of MsbA from E coli, the alpha isomer of dodecylmaltoside (␣-DDM) was used to solubilize and extract MsbA directly from the processed membranes as an intact homodimer, as suggested by gel filtration The bacterial cell wall was removed from the crude cell membranes by freeze-thawing and stirring the cell paste for hours at 4°C in cracking buffer (20 mM Tris pH 7.5, 100 mM NaCl, 20 mM imidazole pH 8.0) containing lysozyme (0.5 mg lϪ1) Following centrifugation at 6000 ϫ g for 40 minutes, the cells were resuspended in lysing buffer (20 mM Tris pH 7.5, 20 mM NaC1) containing DNase I (0.1 mg lϪ1) and stirred at 4°C for hours Cell membranes were then harvested by centrifugation at 6000 ϫ g for 30 minutes and resuspended in an equal volume of solubilization buffer (20 mM Tris pH 7.5, 100 mM NaCl, 30 mM imidazole pH 8.0) containing 1% dodecyl-␣-D-maltoside (␣-DDM, Anatrace, Maumee, WI) for hours The insoluble fraction was separated from the crude detergent-solubilized extract at 29 000 ϫ g for hour The solubilized MsbA protein was purified first by metal affinity chromatography using a column loaded with nickel-NTA superflow resin (Qiagen) or nickel-NTA fast-flow sepharose (Pharmacia Biotech) that was in a low-salt buffer Following an extensive wash, nonspecific binding was reduced with high salt buffer The column was then re-equilibrated with lowsalt buffer and another stringency step was introduced using 60 mM imidazole buffer The MsbA protein, which was still bound to the resin, was eluted using 300 mM imidazole buffer and the resultant fractions combined and diluted fourfold The protein was then loaded on an anion exchange column containing Source 30Q or Source 15Q resin (AmershamPharmacia) Following a brief wash, the protein was eluted by a single high-salt step At this stage, a few minor contaminants were observed and eliminated using a preparative gel filtration chromatography using Superdex 26/60 columns Eco-MsbA runs as a dimer as confirmed by running against protein molecular weight markers by gel filtration chromatography 137 138 ABC PROTEINS: FROM BACTERIA TO MAN Figure 7.2 Crystals of Eco-MsbA grown in 0.05% ␣-DDM A, Two fused crystals, which were split to use in data collection as a native and derivative B, Color change of Eco-MsbA crystal upon uptake of OsCl3, which significantly improved the diffraction quality and resolution The purified MsbA transporter protein was concentrated by a smaller Source 30Q column and then desalted using a HiPrep 26/10 desalting column (Amersham-Pharmacia) into a stabilizing buffer (20 mM Tris pH 7.5, 20 mM NaCl, 0.05% ␣-DDM) and modestly concentrated using a Centricon 100 filtration concentrator (Amicon) prior to freezing aliquots of the protein at Ϫ80°C Final yields of purified MsbA transporter of about 0.5 mg gϪ1 of cell were typical Protein purity was assayed by sodium dodecylsulfate (SDS) gel electrophoresis with Coomassie staining, Western blotting by antihistidine tag antibodies, and monitored using MALDI-TOF (matrix-assisted laser desorption/ ionization – time of flight) mass spectroscopy at the Scripps Center for Mass Spectrometry Samples of MsbA were exceptionally clean with molecular weights within 0.05% of expected PROTEIN CRYSTALLIZATION The crystallization of integral membrane proteins is still a highly empirical process involving a large number of important parameters, which include precipitating reagents, temperature, pH, salt, protein concentration, detergent concentration and detergent type As such, we adopted an approach of screening several detergents and crystallization conditions in parallel using experiments derived from the X-ray structure determination of the mechanosensitive ion channel from Mycobacterium tuberculosis (Chang et al., 1998) After screening and refining approximately 96 000 crystallization conditions for 12 MDR bacterial ABC transporters and detergents, we have grown and analyzed nearly 35 distinct crystal forms At present, the structure of MsbA from E coli (EcoMsbA) in ␣-DDM has been successfully determined Crystals of Eco-msbA were grown at 5°C by the sitting-drop method using protein at approximately 10–15 mg mlϪ1 and a final detergent concentration of 0.05% ␣-DDM (Figure 7.2A) Crystallization trials were performed using a multivariate crystallization matrix of temperatures, detergents and several precipitants The protein was mixed in a ratio of 1:1 or 3:1 with reservoir solution containing 100–200 mM citrate buffer (pH 4.8 to 5.4), 15 to 20% PEG 300, 80–120 mM Li2SO4 or (NH4)2SO4 and 0.05% ␣-DDM Crystals appeared within weeks and continued to grow for months to a full size (0.4 mm ϫ 0.8 mm ϫ 0.3 mm) Gel electrophoresis using SDS–PAGE, Western blotting using anti-histidine tag antibodies, and mass spectroscopy clearly indicated that the crystals were composed of Eco-MsbA To further verify the identity, crystals were washed and dissolved, and the NH2-terminal amino acid sequence was determined to five residues CRYSTALLOGRAPHIC ANALYSIS Crystals of Eco-MsbA using ␣-DDM grew in space group P1 (a ϭ 107.8 Å, b ϭ 126.1 Å, c ϭ 206.6 Å, ␣ ϭ 83.5°, ␤ ϭ 76.3°, ␥ ϭ 84.1°) The native crystals diffracted to a resolution X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA of ϳ6.2 Å using synchrotron radiation at the Stanford Synchrotron Radiation Laboratory (SSRL) but the data was fairly anisotropic In an effort to improve protein lattice contacts and decrease the disorder within the crystals, we applied a crystal refinement strategy that was also used for the structure determination of MscL (Chang et al., 1998), which included the screening of an extensive matrix of detergent types, detergent concentrations, salts, temperatures, organics, additives, deuterium oxide, and heavy metals One compound, OsCl3, greatly improved the diffraction quality to a limiting resolution of 4.5 Å with spots observed to 3.8 Å The binding of OsCl3 made the crystals turn yellowish-brown in color (Figure 7.2B) This heavy atom compound was later found to bind at crystal lattice contacts between the NBDs of two transporters in the unit cell In view of the greatly improved diffraction quality, data collected from this soaked crystal was used as the ‘native’ data set for X-ray structure determination Protein phases were determined by the method of single isomorphous replacement (SIR/SAS) using two evenly split fragments from a single crystal and also by phase combination with a two-wavelength multiple anomalous dispersion (MAD) using the computer package PHASES (Furey and Swaminathan, 1997) Initial electron density maps clearly revealed that the asymmetric unit contained four complete Eco-MsbA transporters (eight monomers) in a pseudo-222 arrangement consistent with the operators of the self-rotation function The Matthews coefficient is 5.0 Å3 per dalton, which is a reasonable value for most membrane protein crystals and corresponds to a solvent/detergent content of ϳ75% Electron density correlations on experimentally derived maps indicated that there were some differences between the transporters that did and did not bind osmium heavy atoms Transporters that did not bind OsCl3 did not adhere to a perfect twofold relating the monomers within a dimer These differences were accommodated in the averaging masks and subsequent density modification procedures Iterative eightfold non-crystallographic symmetry averaging, solvent flattening/flipping, phase extension, and amplitude sharpening using in-house programs yielded electron density maps of excellent quality for tracing the polypeptide chain A chemical model was built using the program CHAIN (Sacks, 1988), and the protein sequence registration was established for bulky aromatic groups in the transmembrane helices from the electron density maps Regions of the histidine ABC domain HisP (Hung et al., 1998) served as a useful guide for modeling the NBD of EcoMsbA (Hung et al., 1998) Numerous rounds of vector refinement were performed using the program XPLOR to best fit the model into the sharpened electron density maps Using this preliminary model, anisotropic corrections to the diffraction intensities were applied using the program XPLOR The crystal structure refinement of Eco-MsbA was complicated by a rapid decrease in the intensity of the diffraction pattern as a function of resolution, corresponding to an overall temperature factor of ϳ150 Å2 Similar temperature factors were reported for the original Kϩ ion channel from Streptomyces lividans (KcsA) as well as for MscL (Chang et al., 1998; Doyle et al., 1998) The experimentally phased electron density maps, however, appeared to be of much higher quality than one would expect for this temperature factor This suggested that there were predominant orientations for the transporters in the crystal that were probably stabilized by heavy-atom binding with additional orientations that introduced a degree of positional disorder As a consequence, whereas the diffraction intensities remained relatively strong at lower resolution, the scattering contributions from this ensemble of transporters and associated detergent interfered at higher resolution, resulting in a rapid decrease in diffraction intensities at a higher resolution Standard crystallographic refinement protocols are generally inadequate for modeling this type of positional disorder and, as a result, we were unable to refine a single model of Eco-msbA to values of R and Rfree below ϳ38 and ϳ45%, respectively In an effort to better simulate the data, we used a multicopy refinement procedure (mc refinement) using 16 copies of the asymmetric unit (eight molecules per asymmetric unit) against the OsCl3 data with very strict eightfold noncrystallographic harmonic constraints (2000 kcal molϪl) between the monomers using a modified version of the program XPLOR (Figure 7.3) (Brunger et al., 1987; Gros et al., 1990; Kuriyan et al., 1991; Pellegrini et al., 1997) After molecular dynamics refinement, an ensemble of very similar models (average root mean square deviation of Ca atoms among models of 1.4 Å) was achieved with a crystallographic R value of 27% and an Rfree of 38% Multicopy refinements were also done using 5, and 10 copies in the asymmetric unit and these mc refinements yielded similar crystallographic R factors and 139 140 ABC PROTEINS: FROM BACTERIA TO MAN STRUCTURE DESCRIPTION Figure 7.3 Stereoviews of 16 superimposed models from multicopy (mc) refinement of Eco-msbA The models are shown in different colors A, View of the dimer looking into the chamber opening B, View of the dimer looking from the lipid bilayer at the external (embedded) surface of the chamber opening Helical regions are more similar while loop regions are generally less ordered B-factors for all atoms were fixed at 90 Å2 during the mc refinement An averaged model was computed and used for analysis Reprinted with permission from G Chang and C.B Roth, Science 293, 1793 (2001) Copyright 2001 American Association for the Advancement of Science Rfree values Most importantly, the significant drop in the Rfree values during the multicopy refinements and the similarity of the independently averaged models suggested a proper fit of the data The choice of using 16 copies of the asymmetric unit provided an opportunity to observe their spatial distribution more finely and was the maximum limit of the computing resources at the time Residue positions in helical regions were very well defined in these models, whereas the loop regions were less ordered, as expected An averaged model with good stereochemistry was computed and used for structural analysis B factors for all atoms were fixed at 90 Å2 during the multicopy refinement The X-ray crystal structure of Eco-MsbA transporter is consistent with the molecule being a homodimer and each subunit is composed of two domains (Figure 7.4) We have identified a third domain bridging the TMD and the NBD Eco-MsbA is approximately 120 Å in length, with the TMD, including the membranespanning region, accounting for approximately 52 Å All the transmembrane ␣-helices are tilted between 30° and 40° from the normal for the membrane, forming a cone-shaped structure with two substantial openings on either side facing the lipid bilayer These openings are approximately 25 Å wide in the longest dimension and lead into a large cone-shaped chamber in the interior of the molecule’s TMD The leaflet half of the membrane domain in the outer membrane leaflet domain forms the inter-molecular contacts holding the two monomers of the transporter together The dimer interface, which is mostly contributed by the second and fifth membrane-spanning ␣-helices, buries approximately 850 Å2 of solvent accessible surface area The base of the chamber facing the cytoplasm is approximately 45 Å in the widest dimension, and the volume of the chamber can easily accommodate lipid A molecules The resolved regions of the NBDs share no intermolecular contact and are separated by approximately 50 Å in the closest dimension Approximately 200 residues are not resolved in this particular crystal form of the molecule, which is free of nucleotide and ligand We assume that this part of the structure is disordered in the absence of either ligand or nucleotide and probably plays an important role in the mechanism of substrate translocation TRANSMEMBRANE DOMAIN STRUCTURE Eco-MsbA begins with the NH2-terminus on the cytoplasmic side as a helix (residue 10–21) that is parallel with the lipid bilayer (Figure 7.4) Residues Trp10, Phe13 and Trp17 would intercalate into the inner leaflet side of the cell membrane The polypeptide chain continues into the first transmembrane helix (TM1, residues 22–52) and leads into the first extracellular loop (EC1, residues 53–64), which crosses X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA Figure 7.4 Structure of Eco-MsbA A, View of the dimer looking into the chamber opening The transmembrane domain, nucleotide-binding domain and intracellular domain are colored red, cyan and dark blue, respectively Transmembrane ␣-helices are marked and the connecting loops are shown in green A model of lipid A (not in the crystal structure) is shown to the right embedded in the lower bilayer leaflet Solid and dotted green lines represent the boundaries of the membrane bilayer leaflets Dotted cyan lines indicate the approximate location of the disordered region in the NBD B, View of Eco-MsbA from the extracellular side, perpendicular to the membrane with a model of lipid A Transporter dimensions are labeled and images were rendered using BOBSCRIPT and RASTER 3D Reprinted with permission from G Chang and C.B Roth, Science 293, 1793 (2001) Copyright 2001 American Association for the Advancement of Science over to the far side of the transporter The electron densities for residues 58–63 of EC1 are diffuse The second transmembrane helix (TM2, residues 65–96) forms part of the opening of the flippase chamber and appears mobile when comparing TM2 ␣-helices from other copies of Eco-MsbA in the unit cell The third and fourth transmembrane helices (TM3, residues 140–164 and TM4, residues 168–192) are connected by a very short extracellular loop (EC2, residues 165–167), which has a polar character Strong electron density is present for several bulky residues, which include Phe161, Tyr162 and Tyr163 in TM3 and Trp165 in EC2 The fifth transmembrane helix (TM5, residues 253–272) begins with a kink caused by Pro253 and forms half of the opening facing the bilayer TM5 is connected to the sixth transmembrane helix (TM6, residues 281–301) by an extracellular loop (EC3, residues 273–280) Although the absolute orientation of Eco-MsbA in the membrane is not known, the putative third extracellular loop (EC3) in human Pgp aligns by sequence homology to Eco-MsbA EC3 and has been shown to be located on the cell surface by in vivo topology studies using the antibody UIC2 (Zhou et al., 1999) TM2 and TM5 141 142 ABC PROTEINS: FROM BACTERIA TO MAN from opposing monomers within the dimer form the major dimerization contact and are well positioned to serve as a hinge allowing the transporter to undergo significant structural rearrangements A EC1 TM5 EC2 EC3 TM1 TM4 TM6 TM3 TM2 NBD STRUCTURE The NBD (colored cyan in Figure 7.4) is the most conserved feature of the ABC transporter family and contains the Walker A/B motif along with the ABC signature motif In the absence of ATP or nucleotide analogue in this crystal form, residues 341–418, which include the Walker A motif, are disordered in our electron density maps The crystal packing, however, suggests sufficient volume to accommodate the mass of the Walker A region The remaining portion of the NBD is well resolved in our structure and includes an ␣-helix (residues 331–340) and residues 418–561, which contain the ABC signature motif (colored pink in Figure 7.5) and the Walker B region (colored gray in Figure 7.5) The NBD of Eco-MsbA has significant similarity in sequence and structure to the corresponding regions found in histidine ABC domain (HisP) with a root mean square (rms) deviation of ϳ1.5 Å (C␣) for residues 445–528 NBD residues that are in direct contact with the intracellular domain (residues 420–448, 500–508 and 531–556) are among some of the most highly conserved regions among all members of the multidrug resistance transporters The COOHterminus of Eco-MsbA (residues 565–582) is not resolved INTRACELLULAR DOMAIN (ICD) STRUCTURE A distinctive feature of the MsbA is two extensive intracellular regions, which we called ICD1 (residues 97–193) and ICD2 (residues 193–252) We have identified a third intracellular subdomain, which connects TM6 with the NBD and which we label ICD3 (residues 302–327) We collectively designate the region located between the TMD and the NBD as the intracellular domain (colored dark blue in Figure 7.4) Most of the residues that correspond to structural elements in ICD1 that are in contact with the NBD are highly conserved throughout multidrug resistance ABC transporters ICD1 (colored brown in Figure 7.5), which is sandwiched between ICD2 (colored violet in Figure 7.5) and the NBD, is composed Cytoplasm NH2 ABC Signature ICD2 ICD1 ICD3 Walker B EC1 B EC2 EC3 TM1 TM6 TM5 TM4 TM3 TM2 Cytoplasm NH2 ICD2 ICD1 ABC Signature ICD3 Walker B Figure 7.5 View of Eco-MsbA looking (A) from the lipid bilayer at the external (embedded) surface of the chamber opening and (B) looking at the interior of the chamber The transmembrane domain is colored red and the transmembrane ␣-helices are marked The NBD is colored cyan with the Walker B motif and ABC signature motif highlighted in gray and pink respectively ICD1, ICD2 and ICD3 are colored brown, violet and yellow respectively for clarity The estimated cell membrane (ϳ 35 Å) and the boundary between the bilayer leaflets are illustrated as solid and dotted yellow lines Figure was rendered using BOBSCRIPT and RASTER 3D Reprinted with permission from G Chang and C.B Roth, Science 293, 1793 (2001) Copyright 2001 American Association for the Advancement of Science of three ␣-helices connected by short loops to form a ‘U’-like structure The second ␣-helix of ICD1 (residues 111–121) is highly conserved and is nestled against residues 420–430 of the NBD The well-ordered portions of ICD2 in our electron density maps are mostly ␣-helical (residues X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA CHAMBER STRUCTURE A TMD Cytoplasm ICD Opening to Chamber NBD B TMD Cytoplasm ICD NBD Figure 7.6 Molecular surface rendering of Eco-MsbA with electrostatic potentials This figure was generated with the program GRASP (54) assuming an ionic strength of 100 mM NaCl and dielectric constant of and 80 for protein and solvent, respectively The surface potential varies continuously from blue (positive) to red (negative) The cell membrane, which is ϳ35 Å wide, is represented with yellow lines The transmembrane domain (TMD), intracellular domain (ICD) and NBD are indicated A, Side view of dimer looking into the chamber The opening of the chamber spans the lower bilayer leaflet B, View of Eco-MsbA monomer looking at the inner surface of the chamber The surface potential within the lower half of the chamber is highly positive due to a clustering of lysine and arginine residues Reprinted with permission from G Chang and C.B Roth, Science 293, 1793 (2001) Copyright 2001 American Association for the Advancement of Science 193–207 and 237–252) The electron densities for residues 208–236, however, are diffuse ICD3 links TM6 and the NBD and forms two ␣-helices connected by short loops (colored yellow in Figure 7.5B) The ␣-helix just preceding the NBD (residues 318–329) is conserved and is in direct contact with both ICD1 and ICD2 The chamber is a symmetric structure that is formed from two Eco-MsbA transmembrane domains and extends along the pseudotwofold axis perpendicular to the cell membrane (Figure 7.6) The chamber has an opening (ϳ25 Å) on either side facing the bilayer, providing free access of substrate from the cytoplasmic leaflet of the lipid bilayer while excluding molecules from the outer leaflet The openings to the chamber are defined by intermolecular interactions between TM2 of one monomer and TM5 of another (Figure 7.6A) Residues 268–273 of TM2 lining the opening of the chamber are partially shielded from the bilayer by TM5 The residues lining the chamber are contributed by all 12 transmembrane ␣-helices and could be highly solvated The inner membrane leaflet side of the chamber contains a cluster of positively charged residues (Arg148, Arg183, Lys187, Arg190, Lys194 and Arg296) (Figure 7.6B), which contrasts the significantly less charged and more hydrophobic environment within the outer membrane leaflet side POSSIBLE FLIPPING MECHANISM The structure of MsbA suggests a mechanism for hydrophobic substrate translocation (Figure 7.7) Studies of human Pgp function indicate that residues lining the proposed chamber opening (TM2, TM5 and TM6) play an important role in substrate recognition (Ambudkar et al., 1999) There is significant evidence indicating a cooperative interaction between the two opposing NBDs (Senior and Bhagat, 1998) The intracellular domains (colored purple in Figure 7.7) bridge the TMD and the NBD serving to couple ATP hydrolysis by the NBD to tertiary arrangements of the transmembrane ␣-helices Upon binding of a lipid A molecule, conformational changes relayed from the transmembrane domain to the intracellular domain stimulate nucleotide binding by the NBD (step in Figure 7.7) Movement of TM2, TM5, TM6 and the two NBDs serves to recruit the substrate and close the chamber (step in Figure 7.7) Binding of ATP by the NBDs may result in a conformational shift that promotes the interaction of the adjacent NBDs The cluster of charges lining the chamber on the inner membrane leaflet side 143 144 ABC PROTEINS: FROM BACTERIA TO MAN ATP ADP ADP ATP Figure 7.7 Model for lipid A transport by Eco-MsbA Stages 1–3 begin at right and proceed clockwise See text for details (1) Lipid A binding, triggering of ATP hydrolysis, and recruitment of substrate to chamber (2) Closure of the chamber and translocation of lipid A Interaction between the two NBDs is possible (3) Opening of the chamber, movement of TM2/TM5, release of lipid A to the outer bilayer leaflet, and nucleotide exchange A small yellow circle and a green rectangle denote the hydrophobic tails and sugar headgroups of lipid A, respectively The cell membrane is represented as a set of two horizontal lines separated by a dash to indicate the separation of bilayer leaflets Blue regions indicate positive charge lining the chamber, and purple regions represent the intracellular domain The gray region on the outer membrane side of the chamber is hydrophobic Red and black arrows show the movement of substrate and the structural changes of MsbA, respectively creates an energetically unfavorable microenvironment for a hydrophobic substrate The asymmetric charge distribution in the interior of the chamber is consistent with the vectored transport of substrate from the inner to the outer membrane leaflet Faced with both the charge and the highly polar contribution of potentially bound solvent, the lipid A molecule ‘flips’ into an energetically more favorable position within the outer membrane leaflet side of the chamber (colored gray in Figure 7.7), where it can form hydrophobic interactions The substrate is now properly orientated to enter the outer bilayer leaflet The flipping of substrate initiates nucleotide hydrolysis by the NBD, causing the chamber to undergo structural rearrangements that enable the expulsion of the substrate to the outer membrane leaflet (step in Figure 7.7) Alternatively, it has been proposed that some drug transporters can extrude their substrates directly into the extracellular medium from the inner leaflet of the bilayer (Bolhuis et al., 1996) This entropically driven ‘flip-flop’ mechanism could account for the unusually broad range of hydrophobic drugs and lipids transported by members of this transporter family Several other variations of this mechanism are plausible and will be refined using biochemical and structural information derived from additional MsbA studies Although we believe that MsbA may not be representative of the ABC transporters that import hydrophilic substrates, there is evidence that suggests human Pgp and other multidrug transporters share transport mechanisms and structural components similar to Eco-MsbA (Higgins and Gottesman, 1992) First, human Pgp and LmrA are believed to be lipid flippases like Eco-MsbA and have been shown to transport (van Helvoort et al., 1996) phosphatidylcholine (Smith et al., 1994) and phosphatidylethanolamine (PE) (Margolles et al., 1999), respectively This suggests a common functional ancestry between the eukaryotic multidrug transporter group and the bacterial lipid flippases Secondly, the size and shape of X-RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA the chamber of Eco-MsbA could accommodate a wide variety of amphipathic molecules, which could explain how human Pgp can extrude such an unusually broad range of substrates The crystal structure of MsbA reveals that it is not a pore through the cell membrane, but a molecular machine scanning the lower bilayer leaflet for substrates, accepting them laterally, and flipping them to the outer membrane leaflet The recent 25 Å cryo-EM reconstruction of the MsbA homologue YvcC from Bacillus subtilis reveals a structural architecture that is consistent with Eco-MsbA solved by X-ray crystallography (Chami et al., 2002) The MsbA model when docked into the EM electron density map suggests that the putative YvcC transmembrane domains form a chamber within the membrane Although they cannot be resolved at this resolution, the 30–40° tilt of all the transmembrane helices is clearly evident Strong electron densities for both the intracellular domain and the NBDs allow the crystal structure of MsbA to be docked into the cryo-EM density with confidence, providing the first evidence of a conserved architecture among the bacterial exporters The X-ray crystal structure of Eco-MsbA provides a first insight into the detailed structure of an ABC transporter, and a foundation for understanding the transport mechanisms of the ABC transporter family Adding a second chapter to the ABC transporter story is the recent 3.5 Å structure of the E coli vitamin B12 importer BtuCD Although BtuCD shares little homology to the MDR class exporters that translocate hydrophobic substrates, the structure reveals a new snapshot of an ABC transporter with contact between the ABC cassettes in a manner much like the Rad50 dimer This finding is significant, for contact between the nucleotide binding domains has been thought necessary to explain the cooperative kinetics of ATP hydrolysis during the transport cycle Interestingly, the ABC cassettes remain in contact in the absence of bound nucleotide, which is not the case with MsbA Similar to MsbA, however, contact between the ABC cassette and the membrane-spanning domain is maintained by the ICD, through a conserved set of residues that the authors name the L-loop This observation reinforces the idea that the ICD domain serves as the bridge coupling ATP hydrolysis by the ABC cassette to rearrangements within TMD The mechanistic model presented by Rees et al proposes that the two ABC cassettes of BtuCD maintain contact throughout the transport cycle, while converting hydrolytic energy to leverage on TMD in a ‘power stroke’ The BtuCD structure presents yet another snapshot of the transport cycle, and suggests a mechanism that may be applied to other ABC importers ACKNOWLEDGMENTS Excerpts are reprinted with permission from Chang and Roth, X-ray structure of an intact ABC-transporter MsbA, Science 293, 1793 Copyright 2001, American Association for the Advancement of Science REFERENCES Ambudkar, S.V., Dey, S., Hrycyna, C.A., Ramachandra, M., Pastan, I and Gottesman, M.M (1999) Biochemical, cellular, 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The X- ray crystal structure of Eco -MsbA provides a first insight into the detailed structure of an ABC transporter, and a foundation for understanding the transport mechanisms of the ABC transporter... functional ancestry between the eukaryotic multidrug transporter group and the bacterial lipid flippases Secondly, the size and shape of X- RAY STRUCTURE OF AN INTACT ABC TRANSPORTER, MSBA the chamber of