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• W2028778570 abstract "•New tools enabled EM visualization of dynamic transporter conformations in lipids•Distinct conformational spectra of two homologous ABC transporters were revealed•P-gp prevails in inward-facing conformations under ATP hydrolysis conditions•MsbA with ATP displays both outward and a continuum of inward-facing conformations ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes. ATP-binding cassette (ABC) exporters are ubiquitously found in all kingdoms of life and their members play significant roles in mediating drug pharmacokinetics and multidrug resistance in the clinic. Significant questions and controversies remain regarding the relevance of their conformations observed in X-ray structures, their structural dynamics, and mechanism of transport. Here, we used single particle electron microscopy (EM) to delineate the entire conformational spectrum of two homologous ABC exporters (bacterial MsbA and mammalian P-glycoprotein) and the influence of nucleotide and substrate binding. Newly developed amphiphiles in complex with lipids that support high protein stability and activity enabled EM visualization of individual complexes in a membrane-mimicking environment. The data provide a comprehensive view of the conformational flexibility of these ABC exporters under various states and demonstrate not only similarities but striking differences between their mechanistic and energetic regulation of conformational changes. ATP-binding cassette (ABC) transporters constitute a large family of integral membrane proteins that utilize the energy of ATP hydrolysis to translocate ions, lipids, nutrients, and drugs across lipid bilayers. Based on the directionality of transport, they are classified as either exporters or importers, with the former found in all living species and the latter reported only in prokaryotic systems (Dassa, 2011Dassa E. Natural history of ABC systems: not only transporters.Essays Biochem. 2011; 50: 19-42Crossref PubMed Google Scholar). Many ABC exporters are promiscuous and bind a wide array of structurally unrelated compounds, in contrast to most importers, which are functionally dependent on peripheral binding proteins for specific substrate recognition (Locher et al., 2002Locher K.P. Lee A.T. Rees D.C. The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism.Science. 2002; 296: 1091-1098Crossref PubMed Scopus (932) Google Scholar, Oldham et al., 2007Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Crystal structure of a catalytic intermediate of the maltose transporter.Nature. 2007; 450: 515-521Crossref PubMed Scopus (420) Google Scholar). ABC exporters are medically important since their members contribute to antibiotic or antifungal resistance of human pathogens, the development of multiple drug resistance (MDR), and several human genetic disorders due to protein dysfunctions. A prominent example is P-glycoprotein (P-gp), which affects the pharmacokinetics of numerous drugs and is implicated in MDR of many human cancers, HIV, and epileptic diseases (Eckford and Sharom, 2009Eckford P.D. Sharom F.J. ABC efflux pump-based resistance to chemotherapy drugs.Chem. Rev. 2009; 109: 2989-3011Crossref PubMed Scopus (481) Google Scholar, Giacomini et al., 2010Giacomini K.M. Huang S.M. Tweedie D.J. Benet L.Z. Brouwer K.L. Chu X. Dahlin A. Evers R. Fischer V. Hillgren K.M. et al.Membrane transporters in drug development.Nat. Rev. Drug Disc. 2010; 9: 215-236Crossref PubMed Scopus (2552) Google Scholar). ABC exporters share a common architecture, including a minimum of two transmembrane domains (TMDs) and two highly conserved nucleotide binding domains (NBDs). The four core domains are commonly either coexpressed as a dimer of TMD-NBD halves, or fused into a single polypeptide chain (Figure S1 available online). The TMDs form the translocation pathway and determine the substrate specificity, whereas the NBDs are thought to associate upon ATP binding and dissociate driven by ATP hydrolysis. The ATP binding and hydrolysis steps are coupled to significant conformational rearrangements of the TMDs opening toward the cytoplasm (also termed inward-facing [IF]) or the periplasm (outward-facing [OF]) (Higgins and Linton, 2004Higgins C.F. Linton K.J. The ATP switch model for ABC transporters.Nat. Struct. Mol. Biol. 2004; 11: 918-926Crossref PubMed Scopus (597) Google Scholar). The alternate access presentation of membrane openings of ABC transporters and other types of membrane pumps has long been used to explain the substrate translocation (Jardetzky, 1966Jardetzky O. Simple allosteric model for membrane pumps.Nature. 1966; 211: 969-970Crossref PubMed Scopus (882) Google Scholar). However, despite a wealth of biochemical and structural data obtained on these transporters from decades of research, many aspects of the translocation process, such as the spectrum of conformational dynamics, the impact of substrate binding, and how the NBD and TMD movements are coupled, remain to be fully elucidated. Previous high-resolution X-ray structural studies revealed large conformational variability within the group of ABC exporters, including prokaryotic MsbA (Ward et al., 2007Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Flexibility in the ABC transporter MsbA: alternating access with a twist.Proc. Natl. Acad. Sci. USA. 2007; 104: 19005-19010Crossref PubMed Scopus (649) Google Scholar), Sav1866 (Dawson and Locher, 2006Dawson R.J. Locher K.P. Structure of a bacterial multidrug ABC transporter.Nature. 2006; 443: 180-185Crossref PubMed Scopus (1083) Google Scholar), TM287/288 (Hohl et al., 2012Hohl M. Briand C. Grutter M.G. Seeger M.A. Crystal structure of a heterodimeric ABC transporter in its inward-facing conformation.Nat. Struct. Mol. Biol. 2012; 19: 395-402Crossref PubMed Scopus (197) Google Scholar, Hohl et al., 2014Hohl M. Hurlimann L.M. Bohm S. Schoppe J. Grutter M.G. Bordignon E. Seeger M.A. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.Proc. Natl. Acad. Sci. USA. 2014; 111: 11025-11030Crossref PubMed Scopus (90) Google Scholar), and eukaryotic P-gp (Aller et al., 2009Aller S.G. Yu J. Ward A. Weng Y. Chittaboina S. Zhuo R. Harrell P.M. Trinh Y.T. Zhang Q. Urbatsch I.L. et al.Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding.Science. 2009; 323: 1718-1722Crossref PubMed Scopus (1629) Google Scholar, Jin et al., 2012Jin M.S. Oldham M.L. Zhang Q. Chen J. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans.Nature. 2012; 490: 566-569Crossref PubMed Scopus (382) Google Scholar, Ward et al., 2013Ward A.B. Szewczyk P. Grimard V. Lee C.W. Martinez L. Doshi R. Caya A. Villaluz M. Pardon E. Cregger C. et al.Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.Proc. Natl. Acad. Sci. USA. 2013; 110: 13386-13391Crossref PubMed Scopus (199) Google Scholar), ABCB10 (Shintre et al., 2013Shintre C.A. Pike A.C. Li Q. Kim J.I. Barr A.J. Goubin S. Shrestha L. Yang J. Berridge G. Ross J. et al.Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states.Proc. Natl. Acad. Sci. USA. 2013; 110: 9710-9715Crossref PubMed Scopus (186) Google Scholar), and ABCB homologs (Kodan et al., 2014Kodan A. Yamaguchi T. Nakatsu T. Sakiyama K. Hipolito C.J. Fujioka A. Hirokane R. Ikeguchi K. Watanabe B. Hiratake J. et al.Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog.Proc. Natl. Acad. Sci. USA. 2014; 111: 4049-4054Crossref PubMed Scopus (138) Google Scholar, Lee et al., 2014Lee J.Y. Yang J.G. Zhitnitsky D. Lewinson O. Rees D.C. Structural basis for heavy metal detoxification by an Atm1-type ABC exporter.Science. 2014; 343: 1133-1136Crossref PubMed Scopus (134) Google Scholar, Srinivasan et al., 2014Srinivasan V. Pierik A.J. Lill R. Crystal structures of nucleotide-free and glutathione-bound mitochondrial ABC transporter Atm1.Science. 2014; 343: 1137-1140Crossref PubMed Scopus (163) Google Scholar) (Figure S1). Notably, most of these structures have been solved in IF states in both the absence and the presence of nucleotide, and a range of amplitudes of the NBD separation has been observed in different species. X-Ray structures of OF states have been obtained for only two prokaryotic proteins with bound nucleotides (Sav1866 and MsbA) (Dawson and Locher, 2006Dawson R.J. Locher K.P. Structure of a bacterial multidrug ABC transporter.Nature. 2006; 443: 180-185Crossref PubMed Scopus (1083) Google Scholar, Ward et al., 2007Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Flexibility in the ABC transporter MsbA: alternating access with a twist.Proc. Natl. Acad. Sci. USA. 2007; 104: 19005-19010Crossref PubMed Scopus (649) Google Scholar). Most recently, a novel nucleotide-bound, occluded outward conformation has been reported for an antibacterial peptide ABC exporter (McjD) (Choudhury et al., 2014Choudhury H.G. Tong Z. Mathavan I. Li Y. Iwata S. Zirah S. Rebuffat S. van Veen H.W. Beis K. Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state.Proc. Natl. Acad. Sci. USA. 2014; 111: 9145-9150Crossref PubMed Scopus (149) Google Scholar). This newly solved structure is proposed as a transition intermediate between previously reported inward-open and outward-open states (Figure S1), providing further steps along the conformational pathway of ABC exporters. The available structures are commonly used as a framework to describe the trajectory of a universal ABC transporter. As the data originate from multiple species, it is not clear to what extent the findings can be generalized (Rees et al., 2009Rees D.C. Johnson E. Lewinson O. ABC transporters: the power to change.Nat. Rev. Mol. Cell Biol. 2009; 10: 218-227Crossref PubMed Scopus (890) Google Scholar). Also, the large NBD separation observed in some IF X-ray structures and its physiological relevance are under much dispute, as this conformation could arise from crystallographic constraints, the use of detergents, or the absence of nucleotides and ligands (Gottesman et al., 2009Gottesman M.M. Ambudkar S.V. Xia D. Structure of a multidrug transporter.Nat. Biotechnol. 2009; 27: 546-547Crossref PubMed Scopus (63) Google Scholar, Jones and George, 2014Jones P.M. George A.M. A reciprocating twin-channel model for ABC transporters.Q. Rev. Biophys. 2014; 47: 189-220Crossref PubMed Scopus (29) Google Scholar). Adding to this complexity, measurements from different methods, including crosslinking (Loo et al., 2010Loo T.W. Bartlett M.C. Clarke D.M. Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.Biochem. Biophys. Res. Commun. 2010; 395: 436-440Crossref PubMed Scopus (54) Google Scholar, Loo and Clarke, 2014Loo T.W. Clarke D.M. Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.J. Biol. Chem. 2014; 289: 8484-8492Crossref PubMed Scopus (18) Google Scholar), fluorescence resonance energy transfer (FRET) or luminescence resonance energy transfer (LRET) (Borbat et al., 2007Borbat P.P. Surendhran K. Bortolus M. Zou P. Freed J.H. McHaourab H.S. Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis.PLoS Biol. 2007; 5: e271Crossref PubMed Scopus (123) Google Scholar, Cooper and Altenberg, 2013Cooper R.S. Altenberg G.A. Association/dissociation of the nucleotide-binding domains of the ATP-binding cassette protein MsbA measured during continuous hydrolysis.J. Biol. Chem. 2013; 288: 20785-20796Crossref PubMed Scopus (35) Google Scholar, Qu and Sharom, 2001Qu Q. Sharom F.J. FRET analysis indicates that the two ATPase active sites of the P-glycoprotein multidrug transporter are closely associated.Biochemistry. 2001; 40: 1413-1422Crossref PubMed Scopus (85) Google Scholar, Verhalen et al., 2012Verhalen B. Ernst S. Borsch M. Wilkens S. Dynamic ligand-induced conformational rearrangements in P-glycoprotein as probed by fluorescence resonance energy transfer spectroscopy.J. Biol. Chem. 2012; 287: 1112-1127Crossref PubMed Scopus (75) Google Scholar), electron paramagnetic resonance (EPR) spectroscopy (Borbat et al., 2007Borbat P.P. Surendhran K. Bortolus M. Zou P. Freed J.H. McHaourab H.S. Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis.PLoS Biol. 2007; 5: e271Crossref PubMed Scopus (123) Google Scholar, van Wonderen et al., 2014van Wonderen J.H. McMahon R.M. O'Mara M.L. McDevitt C.A. Thomson A.J. Kerr I.D. Macmillan F. Callaghan R. The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis.FEBS J. 2014; 281: 2190-2201Crossref PubMed Scopus (33) Google Scholar, Wen et al., 2013Wen P.C. Verhalen B. Wilkens S. McHaourab H.S. Tajkhorshid E. On the origin of large flexibility of P-glycoprotein in the inward-facing state.J. Biol. Chem. 2013; 288: 19211-19220Crossref PubMed Scopus (105) Google Scholar, Zou et al., 2009Zou P. Bortolus M. McHaourab H.S. Conformational cycle of the ABC transporter MsbA in liposomes: detailed analysis using double electron-electron resonance spectroscopy.J. Mol. Biol. 2009; 393: 586-597Crossref PubMed Scopus (123) Google Scholar), or mass spectrometry (Marcoux et al., 2013Marcoux J. Wang S.C. Politis A. Reading E. Ma J. Biggin P.C. Zhou M. Tao H. Zhang Q. Chang G. et al.Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump.Proc. Natl. Acad. Sci. USA. 2013; 110: 9704-9709Crossref PubMed Scopus (136) Google Scholar), are not always in agreement, which has resulted in significant debate in the field. Here, we used single particle electron microscopy (EM) to directly visualize the conformational spectra of two homologous (∼30% sequence identity) ABC exporters: bacterial MsbA (Escherichia coli) and the mammalian P-gp (Mus musculus) (Figure S2). MsbA is a homodimer of TMD-NBD halves and reported to function as a lipid A and phospholipid flippase (Doerrler et al., 2004Doerrler W.T. Gibbons H.S. Raetz C.R. MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli.J. Biol. Chem. 2004; 279: 45102-45109Crossref PubMed Scopus (197) Google Scholar, Zhou et al., 1998Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R. Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis.J. Biol. Chem. 1998; 273: 12466-12475Crossref PubMed Scopus (282) Google Scholar) and also to transport multiple drugs (Eckford and Sharom, 2008Eckford P.D. Sharom F.J. Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates.J. Biol. Chem. 2008; 283: 12840-12850Crossref PubMed Scopus (77) Google Scholar, Reuter et al., 2003Reuter G. Janvilisri T. Venter H. Shahi S. Balakrishnan L. van Veen H.W. The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities.J. Biol. Chem. 2003; 278: 35193-35198Crossref PubMed Scopus (127) Google Scholar). P-gp is a monomer with two pseudosymmetric halves of TMD-NBD fused together by a flexible linker of ∼70 amino acids (Figures S1 and S2) (Ward et al., 2013Ward A.B. Szewczyk P. Grimard V. Lee C.W. Martinez L. Doshi R. Caya A. Villaluz M. Pardon E. Cregger C. et al.Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain.Proc. Natl. Acad. Sci. USA. 2013; 110: 13386-13391Crossref PubMed Scopus (199) Google Scholar). Newly developed amphiphiles (Tao et al., 2013Tao H. Lee S.C. Moeller A. Roy R.S. Siu F.Y. Zimmermann J. Stevens R.C. Potter C.S. Carragher B. Zhang Q. Engineered nanostructured beta-sheet peptides protect membrane proteins.Nat. Methods. 2013; 10: 759-761Crossref PubMed Scopus (95) Google Scholar) in complex with lipids that support high ATPase activity and improve protein stability enabled EM visualization of individual complexes of both transporters in a lipid-bilayer-mimicking environment. EM imaging and analysis using an unbiased approach to 3D model construction was used to delineate the entire conformational spectrum of P-gp and MsbA and the influence of nucleotide and substrate binding. Our analysis reveals striking differences between the two transporters regarding the effect of binding nucleotides and substrates on their conformational changes and the range of NBD separation across the entire structural spectrum. Overall, the data provide a comprehensive view of the conformational flexibility of two homologous ABC transporters and add essential new insights into the mechanistic understanding of these machines. We have recently developed novel β sheet peptide (BP) assemblies for stabilizing integral membrane proteins and demonstrated their advantages in maintaining the function and providing well-defined structures of MsbA for EM-based single particle analysis (Tao et al., 2013Tao H. Lee S.C. Moeller A. Roy R.S. Siu F.Y. Zimmermann J. Stevens R.C. Potter C.S. Carragher B. Zhang Q. Engineered nanostructured beta-sheet peptides protect membrane proteins.Nat. Methods. 2013; 10: 759-761Crossref PubMed Scopus (95) Google Scholar). For detailed conformational analysis in this report, we prepared MsbA and P-gp in BP solutions with exogenous lipids added to compose a bicelle (Zhang et al., 2011Zhang Q. Tao H. Hong W.X. New amphiphiles for membrane protein structural biology.Methods. 2011; 55: 318-323Crossref PubMed Scopus (68) Google Scholar) or nanodisc (Nath et al., 2007Nath A. Atkins W.M. Sligar S.G. Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins.Biochemistry. 2007; 46: 2059-2069Crossref PubMed Scopus (357) Google Scholar) bilayer-mimicking system. Thus, the single protein molecules are surrounded by a thin layer of BP and lipid, more amenable to EM imaging than large micelles or vesicles resulting from commonly used detergent and lipid mixtures. For P-gp, the presence of lipid is a prerequisite for drug-stimulated ATPase activity and enhances its thermostability (Bai et al., 2011Bai J. Swartz D.J. Protasevich I.I. Brouillette C.G. Harrell P.M. Hildebrandt E. Gasser B. Mattanovich D. Ward A. Chang G. et al.A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris.PLoS One. 2011; 6: e22577Crossref PubMed Scopus (78) Google Scholar). The unprocessed EM micrographs and 2D class averages (see below) clearly show individual particles in multiple conformations for both MsbA and P-gp (Figure 1; Figure S3). Comparing samples prepared with and without added exogenous lipids, we note that upon inclusion of lipids, the EM densities near the transmembrane region of MsbA and P-gp appear enlarged, suggesting that lipids preferentially associate with the hydrophobic TMDs as expected (Figures 1A versus 1B, 1C). We further used specific protein labeling on MsbA to unambiguously identify the location of the NBDs and the extracellular site in 2D images (Figure 2). Two single cysteine (Cys) mutants, one in the extracellular loop 2 (Q166C) and the other in the NBD near the cytoplasmic C terminus (T561C), were selected for labeling with biotin C2 maleimide for subsequent binding of the 60 kDa tetrameric NeutrAvidin protein (Hiller et al., 1987Hiller Y. Gershoni J.M. Bayer E.A. Wilchek M. Biotin binding to avidin. Oligosaccharide side chain not required for ligand association.Biochem. J. 1987; 248: 167-171Crossref PubMed Scopus (146) Google Scholar). Using EM, we observed NeutrAvidin binding to either one or two copies of the periplasmic Q166C, or the cytoplasmic T561C, respectively, both in the OF (left panels) or in different IF conformations (middle and right panels) (Figure 2). The data confirm assignments of the TMDs and NBDs in both IF and OF orientations of MsbA in the 2D images. Binding of NeutrAvidin to the T561C NBD greatly diminished ATPase activity, possibly due to impaired NBD mobility and/or steric hindrance of this large domain (Figure S4) and, consistent with the latter, no OF particles labeled with NeutrAvidin were observed for this mutation. For detailed conformational analysis, we have therefore focused our studies on wild-type and unlabeled transporters. To determine the relative population of individual conformations of MsbA and P-gp, we developed a standardized single particle analysis routine, incorporating reference free alignment and classification protocols (Hohn et al., 2007Hohn M. Tang G. Goodyear G. Baldwin P.R. Huang Z. Penczek P.A. Yang C. Glaeser R.M. Adams P.D. Ludtke S.J. SPARX, a new environment for Cryo-EM image processing.J. Struct. Biol. 2007; 157: 47-55Crossref PubMed Scopus (299) Google Scholar, Lander et al., 2009Lander G.C. Stagg S.M. Voss N.R. Cheng A. Fellmann D. Pulokas J. Yoshioka C. Irving C. Mulder A. Lau P.W. et al.Appion: an integrated, database-driven pipeline to facilitate EM image processing.J. Struct. Biol. 2009; 166: 95-102Crossref PubMed Scopus (578) Google Scholar). This approach sorts the isolated particles into homogenous class averages based on similarity. Averaging of aligned particles significantly increases the signal to noise ratio of each class compared with individual particles facilitating interpretation of the conformational state. To distinguish between different conformations versus relative orientations, we also used a tilt pair approach (random conical tilt [RCT] [Radermacher et al., 1987Radermacher M. Wagenknecht T. Verschoor A. Frank J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli.J. Microsc. 1987; 146: 113-136Crossref PubMed Scopus (427) Google Scholar]) to reconstruct 3D maps. RCT is an established procedure that uses alignment and classification parameters of untilted particle images to determine the relative orientation of their respective partners in the paired tilted particle image and thus directly reconstruct a 3D model of an individual particle class, requiring no a priori information about the particle conformation. This method has been shown to be well suited to the characterization of small flexible proteins (Campbell et al., 2014Campbell M.G. Underbakke E.S. Potter C.S. Carragher B. Marletta M.A. Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase.Proc. Natl. Acad. Sci. USA. 2014; 111: 2960-2965Crossref PubMed Scopus (50) Google Scholar, Chen et al., 2010Chen X. Xie C. Nishida N. Li Z. Walz T. Springer T.A. Requirement of open headpiece conformation for activation of leukocyte integrin alphaXbeta2.Proc. Natl. Acad. Sci. USA. 2010; 107: 14727-14732Crossref PubMed Scopus (86) Google Scholar, Lyumkis et al., 2013Lyumkis D. Doamekpor S.K. Bengtson M.H. Lee J.W. Toro T.B. Petroski M.D. Lima C.D. Potter C.S. Carragher B. Joazeiro C.A. Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase.Proc. Natl. Acad. Sci. USA. 2013; 110: 1702-1707Crossref PubMed Scopus (32) Google Scholar). From multiple 3D maps (25–40 Å resolution range) generated from the RCT analysis, we determined the inter-NBD separation by comparison with a reference library of models created by linear interpolation of the atomic coordinates between the intermediate structures solved by crystallography (Ward et al., 2007Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Flexibility in the ABC transporter MsbA: alternating access with a twist.Proc. Natl. Acad. Sci. USA. 2007; 104: 19005-19010Crossref PubMed Scopus (649) Google Scholar) (Figures 3 and 4). The fit of the EM volume maps to the X-ray structure-derived models verifies that, to the resolution of interest in interpreting these structures, there are no significant structural artifacts introduced by the EM methods used here.Figure 4Conformational Spectra of IF MsbA and P-gpShow full caption(A) X-Ray structures of MsbA and P-gp previously solved in various IF conformations. The color scheme is the same as in Figure S1. Distances between T561 Cα positions of E. coli MsbA and its equivalent positions (marked by red dots) in Vibrio cholerae MsbA and P-gp structures are shown above the PDB ID code. 3G61 included two P-gp molecules solved with slightly different NBD separations.(B) Surface representations of reference models created by linear interpolation of MsbA X-ray structures that are used to categorize the NBD spacings in the structures displayed in (C) and (D).(C) First row shows experimental IF 3D densities from APO MsbA arranged by ascending NBD separation using the distance between T561 as an internal standard. Second row shows superposition of EM density onto X-ray models to emphasize the correspondence between the two. Differences in densities surrounding the TMD come from the detergent/lipid.(D) Experimental maps of P-gp (first row) and superposition onto the same reference library (second row) shown in (B).(E) Statistical occurrence of particles contributing to individual IF categories for nucleotide-free, APO MsbA (light blue), ATP-bound MsbA (dark blue), and nucleotide-free P-gp (orange). For MsbA, the entire conformational spectrum is occupied, and no significant differences in individual IF distributions within the IF population are observed between the APO and ATP sample. The spectrum of P-gp conformations is clustered in the region representing NBD separation between 50 and 75 Å; no structures outside this range are detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) X-Ray structures of MsbA and P-gp previously solved in various IF conformations. The color scheme is the same as in Figure S1. Distances between T561 Cα positions of E. coli MsbA and its equivalent positions (marked by red dots) in Vibrio cholerae MsbA and P-gp structures are shown above the PDB ID code. 3G61 included two P-gp molecules solved with slightly different NBD separations. (B) Surface representations of reference models created by linear interpolation of MsbA X-ray structures that are used to categorize the NBD spacings in the structures displayed in (C) and (D). (C) First row shows experimental IF 3D densities from APO MsbA arranged by ascending NBD separation using the distance between T561 as an internal standard. Second row shows superposition of EM density onto X-ray models to emphasize the correspondence between the two. Differences in densities surrounding the TMD come from the detergent/lipid. (D) Experimental maps of P-gp (first row) and superposition onto the same reference library (second row) shown in (B). (E) Statistical occurrence of particles contributing to individual IF categories for nucleotide-free, APO MsbA (light blue), ATP-bound MsbA (dark blue), and nucleotide-free P-gp (orange). For MsbA, the entire conformational spectrum is occupied, and no significant differences in individual IF distributions within the IF population are observed between the APO and ATP sample. The spectrum of P-gp conformations is clustered in the region representing NBD separation between 50 and 75 Å; no structures outside this range are detected. The OF particles assume a characteristic arrowhead shape in the 2D class averages, readily distinguishable from dissociated NBD shapes of IF particles (Figure 1). The OF particles are confirmed by examination of the 3D density maps corresponding to the 2D class averages (Figure 3) and comparison of the 3D EM maps with the published X-ray structures (Choudhury et al., 2014Choudhury H.G. Tong Z. Mathavan I. Li Y. Iwata S. Zirah S. Rebuffat S. van Veen H.W. Beis K. Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state.Proc. Natl. Acad. Sci. USA. 2014; 111: 9145-9150Crossref PubMed Scopus (149) Google Scholar, Dawson and Locher, 2006Dawson R.J. Locher K.P. Structure of a bacterial multidrug ABC transporter.Nature. 2006; 443: 180-185Crossref PubMed Scopus (1083) Google Scholar, Ward et al., 2007Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Flexibility in the ABC transporter MsbA: alternating access with a twist.Proc. Natl. Acad. Sci. USA. 2007; 104: 19005-19010Crossref PubMed Scopus (649) Google Scholar). Because a side view image of the ABC transporter in IF conformations, rotated by 90°, might have an appearance similar to the OF conformation, we cannot generally exclude the possibility of confusing different views and different conformers. However, our 3D analysis of arrowhead-shaped particles always corresponded to OF conformations and never to IF conformations of the transporters. Of note, our OF EM models cannot distinguish between the outward-open (shown in MsbA and Sav1866) and outward-occluded (M" @default.
• W2028778570 created "2016-06-24" @default.
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• W2028778570 date "2015-03-01" @default.
• W2028778570 modified "2023-10-16" @default.
• W2028778570 title "Distinct Conformational Spectrum of Homologous Multidrug ABC Transporters" @default.
• W2028778570 cites W1487573233 @default.
• W2028778570 cites W1788023629 @default.
• W2028778570 cites W1966516959 @default.
• W2028778570 cites W1967605219 @default.
• W2028778570 cites W1972858806 @default.
• W2028778570 cites W1976251743 @default.
• W2028778570 cites W1978612710 @default.
• W2028778570 cites W1983518163 @default.
• W2028778570 cites W1983751391 @default.
• W2028778570 cites W1985337611 @default.
• W2028778570 cites W1987641460 @default.
• W2028778570 cites W1991504728 @default.
• W2028778570 cites W1991955360 @default.
• W2028778570 cites W1994348363 @default.
• W2028778570 cites W1996360948 @default.
• W2028778570 cites W1997432723 @default.
• W2028778570 cites W2003617902 @default.
• W2028778570 cites W2005046011 @default.
• W2028778570 cites W2005906765 @default.
• W2028778570 cites W2006507421 @default.
• W2028778570 cites W2008037560 @default.
• W2028778570 cites W2010398938 @default.
• W2028778570 cites W2011339029 @default.
• W2028778570 cites W2018031638 @default.
• W2028778570 cites W2022242801 @default.
• W2028778570 cites W2023239354 @default.
• W2028778570 cites W2025867071 @default.
• W2028778570 cites W2028470175 @default.
• W2028778570 cites W2031487956 @default.
• W2028778570 cites W2035506170 @default.
• W2028778570 cites W2035982659 @default.
• W2028778570 cites W2039151526 @default.
• W2028778570 cites W2040855441 @default.
• W2028778570 cites W2041010935 @default.
• W2028778570 cites W2043064789 @default.
• W2028778570 cites W2048982317 @default.
• W2028778570 cites W2052880175 @default.
• W2028778570 cites W2055042191 @default.
• W2028778570 cites W2055625563 @default.
• W2028778570 cites W2061600887 @default.
• W2028778570 cites W2062598925 @default.
• W2028778570 cites W2064697520 @default.
• W2028778570 cites W2066278397 @default.
• W2028778570 cites W2069130875 @default.
• W2028778570 cites W2073949924 @default.
• W2028778570 cites W2076449818 @default.
• W2028778570 cites W2079197876 @default.
• W2028778570 cites W2079301800 @default.
• W2028778570 cites W2084979694 @default.
• W2028778570 cites W2087944803 @default.
• W2028778570 cites W2092435069 @default.
• W2028778570 cites W2094763327 @default.
• W2028778570 cites W2095182463 @default.
• W2028778570 cites W2108047993 @default.
• W2028778570 cites W2111935912 @default.
• W2028778570 cites W2119981825 @default.
• W2028778570 cites W2132629607 @default.
• W2028778570 cites W2134193705 @default.
• W2028778570 cites W2134795955 @default.
• W2028778570 cites W2135228046 @default.
• W2028778570 cites W2139093124 @default.
• W2028778570 cites W2143859034 @default.
• W2028778570 cites W2146142858 @default.
• W2028778570 cites W2150246575 @default.
• W2028778570 cites W2157393167 @default.
• W2028778570 cites W2160899469 @default.
• W2028778570 cites W2161661741 @default.
• W2028778570 cites W2161983628 @default.
• W2028778570 cites W2166189591 @default.
• W2028778570 cites W2171310392 @default.
• W2028778570 cites W2324172260 @default.
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