Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB
The AcrA-AcrB-TolC complex is a secondary active antiport system in Escherichia coli , powered by the electrochemical proton gradient across the inner membrane of this Gram-negative bacterium ( Okusu et al . , 1996 ) . Its biological function is to capture cytotoxic compounds from either the periplasmic space or the inner membrane , and to expel them across the outer membrane , into the extracellular medium . This tripartite system is arguably one of the most complex and intriguing secondary transporters known to date , from both structural and mechanistic standpoints . The AcrAB-TolC system and its homologues are also of considerable biomedical interest , as they confer multidrug resistance to important pathogenic bacteria involved in human disease ( Nikaido and Pagès , 2012 ) . AcrB is the inner-membrane component of the system , and is responsible for both substrate recognition and energy transduction . This membrane protein belongs to the resistance-nodulation-cell-division ( RND ) family ( Tseng et al . , 1999 ) , which also includes transporters involved in protein secretion ( e . g . , SecDF of E . coli ) , export of lipids ( e . g . , MmpL7 of Mycobacterium tuberculosis ) and pigment export ( e . g . , ORF4 of Xanthomonas oryzae ) . In humans , the RND transporters NPC1 and Patched play key roles in the regulation of cholesterol metabolism and in the Hedgehog signaling pathway of embryonic cells , respectively ( Taipale et al . , 2002; Scott and Ioannou , 2004 ) . The first known structure of AcrB , solved by X-ray crystallography at a resolution of 3 . 5 Å , revealed a homotrimeric assembly in which all protomers adopt the same conformation ( Murakami et al . , 2002 ) . In each protomer , the transmembrane ( TM ) domain consists of 12 α-helices ( TM1–TM12 ) , mostly interconnected by short unstructured loops as is common . In contrast to other secondary transporters , however , AcrB also features two large periplasmic insertions , between TM1 and TM2 , and between TM7 and TM8 , which account for approximately 60% of the 1049 amino acids in each protomer . These insertions are highly structured domains that project 70 Å away from the membrane , and form extensive interfaces among protomers ( Figure 1 ) . The TM domains barely contribute to the trimer interface , in further contrast to typical homomeric transporters . The more distal region of the periplasmic domain features a central funnel-shaped cavity that channels the substrate towards TolC , the outer-membrane channel component ( 140 Å in length ) of this transport system ( Koronakis et al . , 2000 ) . AcrA , the third component , functions as a peripheral scaffold to mediate the interaction between AcrB and TolC ( Akama et al . , 2004; Mikolosko et al . , 2006; Tikhonova et al . , 2011; Du et al . , 2014 ) . 10 . 7554/eLife . 03145 . 003Figure 1 . Topology and secondary structure representations of the AcrB protomers . ( A ) Topology of the AcrB protomers in the transmembrane ( TM ) and porter domains . Our analysis indicates that the TM domain consists of two 5-helix parallel repeats , referred to as R1 and R2 , and two flanking helices , TM2 and TM8 . R1 ( blue ) is connected to R2 ( red ) through helix Iα ( white ) , which lies parallel to the cytoplasmic face of the membrane . The porter domain also consists of two repeats of two subdomains each , namely PC1 and PN2 ( blue ) , and PN1 and PC2 ( red ) . TM2 ( cyan ) connects R1 to PN2 , whereas TM8 ( orange ) links R2 to PC2 . Other connections among porter sub-domains and R1/R2 are indicated by open/closed squares/circles . Hexagons indicate the connections between the porter domain and the funnel domain ( FD ) , not drawn in this scheme . ( B ) Cartoon representation of the secondary structure of an AcrB protomer , in the context of the complete trimer . The other two protomers are drawn as a molecular surface ( gray ) . Different sub-domains are colored as in ( A ) , with inter-connecting loops and the FD shown in black . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 00310 . 7554/eLife . 03145 . 004Figure 1—figure supplement 1 . Cartoon representation of the porter domain of AcrB in the asymmetric state , viewed from the outer membrane , along the perpendicular to the inner membrane . Each of the protomers adopt a distinct conformation , namely L , T and O; gray dashed lines indicate the protomer interfaces . Subdomains PC1/PN2 and PN1/PC2 are colored in blue and red , respectively , as in Figure 1 . A minocycline molecule is bound in the T state , in the deep binding pocket between PC1 and PN2 . Substrates can also bind more superficially , in the L state . Black dashed lines indicate access pathways from the periplasmic space into the drug-binding sites , in the L and T states , and from the binding site to the central funnel ( green dashed line ) within the funnel domain , in the O state . A fragment of this funnel domain is shown with green cartoons; this funnel is open to the lumen of the TolC channel , which traverses the outer membrane of E . coli . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 00410 . 7554/eLife . 03145 . 005Figure 1—figure supplement 2 . Molecular simulation systems employed in this study . ( A ) Asymmetric AcrB trimer ( residues 1 to 1044 per protomer ) with one minocycline bound to the T state , embedded in a POPC lipid membrane and solvated by water . The system is enclosed in a periodic box of dimensions ∼150 × 150 × 160 Å . The molecular system includes ∼370 , 000 atoms . AcrB is shown as a van-der-Waals surface , with the L , T and O states in blue , yellow and red , respectively . Lipid and water molecules are represented with lines and sticks; free Na+ ions , added to neutralize the total charge of the system , are shown as green spheres . ( B ) Free-energy simulations were carried out on a construct of the individual AcrB protomers , comprising the transmembrane and porter domains only , embedded in a POPC lipid membrane . The figure shows the T state; L and O states were prepared similarly . Each of these systems consist of ∼101 , 000 atoms , enclosed in a rectangular box of dimensions ∼90 × 90 × 120 Å . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 005 The intricate and unusual topology of AcrB and its trimerization state were confirmed by subsequent higher-resolution X-ray structures obtained from crystals grown in different space groups , several of which include bound substrates ( Murakami et al . , 2006; Seeger et al . , 2006; Sennhauser et al . , 2007; Nakashima et al . , 2011 , 2013; Eicher et al . , 2012 ) . These structures , however , also revealed that upon substrate loading AcrB becomes asymmetric , that is , each protomer in the trimer adopts one of three distinct conformational states , designated as ‘loose’ ( L ) , ‘tight’ ( T ) and ‘open’ ( O ) , or alternatively , as ‘access’ , ‘binding’ and ‘extrusion’ , ( Murakami et al . , 2006; Seeger et al . , 2006 ) . On the basis of these structures as well as other findings ( Seeger et al . , 2008; Takatsuka and Nikaido , 2009 ) , it has also become generally accepted that drug recognition and efflux are mediated by a sub-domain of the periplasmic region , referred to as the porter domain ( Figure 1 , Figure 1—figure supplement 1 ) , whose mechanism is akin to that of a peristaltic pump ( Seeger et al . , 2006 ) . Specifically , recognition of high molecular-mass drugs occurs in the L state , via an access pocket in the porter domain that is open to the periplasmic space ( Nakashima et al . , 2011; Eicher et al . , 2012 ) . When the L protomer transitions to the T state , drugs translocate to a binding pocket deeper in the protein; low molecular-mass drugs , however , can apparently bind directly to the deep binding pocket in the T protomer without binding to the access pocket first ( Nakashima et al . , 2011 ) . Drug extrusion occurs next , in the transition from T to O , which re-shapes and opens the binding pocket to a central cavity in the funnel domain , which leads to the lumen of the TolC channel . Finally , the O to L transition resets the conformational cycle by re-exposing the binding site to the periplasm . The extensive interface between protomers in the porter domain , and the asymmetric nature of the trimer , also suggest that the three protomers are conformationally coupled ( Pos , 2009 ) . Indeed , recent measurements of AcrB transport kinetics ( Nagano and Nikaido , 2009; Lim and Nikaido , 2010 ) are consistent with a bi-site activation model ( Pos , 2009 ) , analogous to the binding-change mechanism of the ATP synthase ( Boyer , 1997 ) . The transport mechanism of the TM domain of AcrB , in contrast , remains largely undefined . The asymmetric trimer structures indicate that this domain also cycles between three distinct conformations , and point to several ionizable residues in its core ( D407 , D408 , K940 and R971 ) that might function as a proton-relay network . Nevertheless , no clear interpretation of existing data has been formulated yet in terms of an alternating-access proton transport mechanism , which is a necessary condition for the transporter to harness the energy stored in the transmembrane electrochemical gradient . How this proton-motive-force is mechanically transduced to the porter domain , in order to power uphill drug efflux , also remains to be determined . Here , we gain insights into these crucial mechanistic questions from a series of new crystal structures of AcrB , systematically analyzed with molecular simulations and other computational methods . More specifically , we examine the TM domain of the transporter in an improved atomic structure of wildtype AcrB ( Eicher et al . , 2012 ) , at 1 . 9 Å resolution , and also report the structures of four single-substitution variants ( D407N , D408N , K940A and R971A ) , all of which are inactive both in proton translocation and drug efflux , solved at resolutions between 2 . 0 and 2 . 3 Å . Taken together with our computational analysis , these data enable us to delineate the alternating-access mechanism of proton transport of the TM domain , and rationalize how this mechanism energizes drug efflux , more than 50 Å away .
Despite the unique topological complexity of AcrB , a systematic analysis of its structure permits to rationalize its conformational cycle in terms of collective motions of distinct structural units . In the porter domain , as noted previously , each protomer consists of two structural repeats , referred to as PN1/PC2 , and PN2/PC1 ( Figure 1 , Figure 1—figure supplement 1 ) . Each repeat is a tandem of two similar α/β sub-domains , joined by a shared β-strand . The PN1/PC2 repeat appears to be a rigid unit throughout the LTO conformational cycle . Analysis of the conformation of this repeat in the 1 . 9 Å-resolution crystal structure of wildtype AcrB ( Eicher et al . , 2012 ) yields root-mean-square differences ( RMSD ) among states of less than 0 . 5 Å ( Supplementary file 1 ) . By contrast , the PN2/PC1 tandem changes its conformation significantly , in particular in the L to T ( RMSD ∼2 . 0 Å ) and T to O transitions ( RMSD ∼1 . 8 Å ) . This is due to the rearrangement of PN2 relative to PC1 , rather than to changes in the internal structure of the individual α/β domains , which appear to be quite inflexible across the cycle , in both repeats ( Supplementary file 1 ) . As described elsewhere ( Murakami et al . , 2006; Seeger et al . , 2006; Sennhauser et al . , 2007; Pos , 2009; Nakashima et al . , 2013 ) , the changes in PN2/PC1 reflect binding of the substrate deep within the hydrophobic core of the tandem , in the T state , and its subsequent release into the interior of the funnel domain , in the O state ( Figure 1—figure supplement 1; Video 1 , Video 2 , Video 3 ) . Incidentally , the conformation of the funnel domain is largely unchanged throughout the cycle ( RMSD < 0 . 5 Å ) . 10 . 7554/eLife . 03145 . 006Video 1 . Structural repeats within the periplasmic porter domain of AcrB , PN1/PC2 and PN2/PC1 , and their collective motions during the transport cycle . L to T transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 00610 . 7554/eLife . 03145 . 007Video 2 . Structural repeats within the periplasmic porter domain of AcrB , PN1/PC2 and PN2/PC1 , and their collective motions during the transport cycle . T to O transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 00710 . 7554/eLife . 03145 . 008Video 3 . Structural repeats within the periplasmic porter domain of AcrB , PN1/PC2 and PN2/PC1 , and their collective motions during the transport cycle . O to L transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 008 Given the modular nature of the porter domain , we reasoned that the TM domain might also consist of structural repeats , as is observed in other secondary transporters . To identify these , we quantified the degree of similarity of combinative structural superpositions of fragments of the transmembrane 12-helix bundle in the L , T and O states ( Figure 2 ) . This analysis indicates that the conformational cycle of the TM domain entails relative motions of two structural repeats within each protomer , which had not been previously recognized . Each of these repeats , which we refer to as R1 and R2 , comprise five transmembrane α−helices . The N-terminal repeat , R1 , includes TM1 and TM3 to TM6 , while the C-terminal repeat , R2 , comprises TM7 and TM9 to TM12 ( Figure 1 ) . R1 and R2 are thus oriented in parallel , and are rotated relative to each other approximately 180° around an axis perpendicular to the membrane plane . Interestingly , each repeat is flanked by a single transmembrane helix that seems to function as a coupling element with the periplasmic porter domain ( Figure 1 ) . The flanking helices are TM2 and TM8 , respectively; TM2 is linked to the flexible PN2/PC1 repeat in the porter domain , while TM8 is connected to the more rigid PN1/PC2 unit ( Figure 1 ) . 10 . 7554/eLife . 03145 . 009Figure 2 . Identification of structural units within the TM domain of AcrB undergoing collective motions throughout the LTO conformational cycle . We considered all possible combinations of 6 , 5 and 4 transmembrane helices , within either TM1-TM6 or TM7-TM12 , and compared the structure of each set of helices in the L vs T states ( closed circles ) , T vs O ( open circles ) and O vs L ( closed squares ) , through pairwise root-mean-square difference ( RMSD ) calculations . When these RMSD values are used as a rank , it becomes clear that combinations that exclude TM2 or TM8 ( red ) feature systematically smaller differences across the LTO cycle than those that include TM2 or TM8 ( black ) . All other exclusions show no clear correlation . For example , comparing TM1-TM6 ( left panel ) in L vs T , this increasing ranking is ΔTM2 , ΔTM4 , ΔTM1 , ΔTM5 , ΔTM3 , and ΔTM6; in T vs O: ΔTM2 , ΔTM5 , ΔTM4 , ΔTM3 , ΔTM1 , and ΔTM6; and in O vs L: ΔTM2 , ΔTM5 , ΔTM1 , ΔTM4 , ΔTM3 , and ΔTM6 . Furthermore , the data shows that combinations of five helices excluding TM2 and TM8 are not significantly more divergent across the LTO conformers than combinations of four helices . Thus , the asymmetric crystal structure of AcrB captures collective displacements of two 5-helix bundles within each protomer , formed by TM1/TM3-TM6 and TM7/TM9-TM12 ( referred to as repeats R1 and R2 ) , relative to flanking helices TM2 and TM8 , respectively . Note that comparisons of combinations of seven or more helices result in larger RMSD values than those shown here , and are therefore omitted . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 00910 . 7554/eLife . 03145 . 010Figure 2—figure supplement 1 . Structural variations in the R1 and R2 repeats of the transmembrane domain of wildtype AcrB , in the L , T and O states . The off-diagonal elements are comparisons ( in terms of RMSD , as in Supplementary file 1 ) of the same repeat in different conformational states of the AcrB protomer . The diagonal elements are comparisons between R1 and R2 in the same state ( all values in Å ) . Both R1 and R2 consist of a five-helix bundle . R1 includes TM1 and TM3 to TM6 , while R2 comprises TM7 and TM9 to TM12 . The definition of each transmembrane segment is as follows: TM1 , residues 10–28; TM2 , 329–358; TM3 , 362–386; TM4 , 393–423; TM5 , 428–457; TM6 , 463–496 , TM7 , 539–557; TM8 , 872–892; TM9 , 895–919; TM10 , 925–955; TM11 , 960–989; TM12 , 998–1031 . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 01010 . 7554/eLife . 03145 . 011Figure 2—figure supplement 2 . Relative displacements of structural elements in the transmembrane and porter domain of wildtype AcrB across the LTO conformational cycle . In each case , the L , T and O states are compared pair-wise . For example , to compute the displacement of R2 between the L and T states , the structures of R1+R2 in these two states are overlaid by least-square fitting R1 , and the RMSD of R2 is computed ( all values in Å ) . An analogous procedure is followed for TM2 , relative to R1 , and TM8 , relative to R2 , as well as for repeat PN1+PC2 in the porter domain relative to PN2+PC1 . In all cases it is apparent that the computed displacements are significantly greater than the changes in the internal structure of these elements , quantified in Supplementary file 1 and Figure 2—figure supplement 1 , indicating collective motions take place . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 011 Analysis of R1 , for example , shows that the structure of this five-helix bundle is largely preserved in the L , T and O conformations; the RMSD among these states ranges from 0 . 8 to 1 . 1 Å ( Figure 2—figure supplement 1 ) . These differences are even smaller for R2 , with RMSD values between 0 . 3 and 1 . 0 Å ( Figure 2—figure supplement 1 ) . R1 and R2 are , however , always different from each other ( RMSD ∼3 . 5 Å ) . Further inspection also shows that R1 and R2 move relative to each other throughout the conformational cycle of each protomer ( Figure 2—figure supplement 2 ) . For example , if we superimpose R1 in the L and T conformations , the RMSD of the corresponding R2 repeats is 1 . 6 Å; for the T to O transition the analogous value is 2 . 1 Å , and for O to L , 3 . 1 Å . These relative differences are substantially larger than those observed individually within R1 or R2 , for all steps of the cycle . Therefore , it is clear that R2 and R1 undergo collective motions during the LTO cycle that are larger in magnitude than the changes in their internal structure . Structural overlays and interpolations of the L , T and O conformations , based on the topological decomposition just described , reveal the nature of the relative repeat motions within the TM domain during the transport cycle ( Figure 3; Video 4 , Video 5 , Video 6 ) . In the L to T transition , R1 and R2 undergo a rocking motion around an axis that is tilted relative to the membrane plane ( Figure 3; Video 4 ) . By contrast , in the transition between the T and O states , and between O and L , what can be observed is mostly a lateral shear motion of R1 and R2 , relative to each other ( Figure 3; Video 5 , Video 6 ) . This analysis also highlights the motions in the helices flanking the repeats , TM2 and TM8 . In the L to T transition , TM2 moves downwards relative to R1 ( toward the cytoplasm ) , returning to the up state in the O conformation ( Figure 3; Video 4 , Video 5 , Video 6 ) . By contrast , TM8 is similarly positioned in the L and T states , and largely unengaged from R2 . However , during the T to O transition this α-helix becomes closely packed against R2 , through hydrophobic interactions with TM5 . In this transition TM8 also becomes elongated , reaching directly to the PN1/PC2 unit ( Figure 3; Video 4 , Video 5 , Video 6 ) . 10 . 7554/eLife . 03145 . 012Figure 3 . Collective motions of the structural repeats in TM domain of AcrB . For clarity , the figure shows only the transmembrane α-helices at the interface between repeats R1 ( blue ) and R2 ( red ) , alongside the two α-helices flanking these repeats , TM2 ( cyan ) , TM8 ( orange ) , and the helix that links the two repeats , Iα ( gray ) . Each panel depicts two conformations in the LTO cycle , overlaid optimally on TM4-TM6; thus the figure highlights the motions of R2 relative to R1 . Initial and final states ( e . g . , L vs T ) are shown in light and bright colors , respectively . Note that the L vs T overlay is viewed from a different angle than T vs O and O vs L , for clarity . Interpolations between these conformational states using the complete TM domain are shown in Video 4 , Video 5 and Video 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 01210 . 7554/eLife . 03145 . 013Figure 3—figure supplement 1 . Arrangement of helix Iα across the conformational cycle of wildtype AcrB . Iα is an additional α-helix inserted between TM6 and TM7 , which lies parallel to the membrane domain ( residues 512 to 536 ) . RMSD analysis ( shown in Å ) of R2+Iα indicates that this helix moves alongside the R2 repeat . RMSD analysis of TM2+I also shows that the relative orientation of these two helices is also approximately constant in the L , T , and O states . Altogether , these data indicate that Iα functions as a connecting rod coupling the displacements of TM2 and R2 . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 01310 . 7554/eLife . 03145 . 014Video 4 . Structural repeats within the transmembrane domain of AcrB , R1 and R2 , and their collective motions during the transport cycle . L to T transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 01410 . 7554/eLife . 03145 . 015Video 5 . Structural repeats within the transmembrane domain of AcrB , R1 and R2 , and their collective motions during the transport cycle . T to O transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 01510 . 7554/eLife . 03145 . 016Video 6 . Structural repeats within the transmembrane domain of AcrB , R1 and R2 , and their collective motions during the transport cycle . O to L transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 016 Interestingly , the membrane domain features an additional helix , Iα , which lies parallel to the membrane , on the cytoplasmic side . This helix is inserted between TM6 and TM7 in the protein sequence , and thus links R1 and R2 ( Figure 1 ) . Structurally , however , Iα is mostly adjacent to R2 , and interacts with R1 ( via hydrophobic interactions and hydrogen-bonds ) primarily at the cytoplasmic end of TM2 , at a right angle ( Figure 3; Video 4 , Video 5 , Video 6 ) . Our analysis shows that Iα is effectively part of R2 , and that it moves rigidly along with this repeat in the three conformational transitions , most probably in response to the motion of TM2 ( Figure 3—figure supplement 1 ) . This notion is consistent with the finding that MexB , a homolog of AcrB , is functional if the mexB gene is expressed in two halves , split at the N-terminal end of Iα ( Eda et al . , 2003 ) . This construct lacks a direct link between Iα and TM6 in R1 , but likely preserves the large interface between Iα and R2 , as well as the interaction with TM2 . Previous crystal structures of AcrB ( Murakami et al . , 2006; Seeger et al . , 2006; Sennhauser et al . , 2007; Eicher et al . , 2012 ) have revealed an intriguing network of ionic and polar side-chains in the core of the TM domain , which seems to re-configure as the TM domain cycles between conformations . In particular D407 and D408 in TM4 and K940 in TM10 are critical for function ( Guan and Nakae , 2001; Murakami and Yamaguchi , 2003; Takatsuka and Nikaido , 2006; Seeger et al . , 2009 ) . Another functionally important side-chain , R971 in TM11 , is located about 10 Å away from K940 on TM10 , towards the cytoplasm . Interestingly , all these side chains are found at the interface between R1 and R2 , seemingly controlling the relative position of the repeats . The latest high-resolution ( 1 . 9 Å ) crystal structure of AcrB ( PDB entry 4DX5 , Eicher et al . , 2012 ) , whose TM domain had not been previously analyzed , reveals in detail how this side-chain network reorganizes throughout the conformational cycle ( Figure 4 ) . In both the L and T states , R1 and R2 appear to be strongly paired through multiple interactions . First , through K940 , which projects away from R2 and is sandwiched between D407 and D408 , from R1 , forming two simultaneous ionic interactions . D407 is the acceptor of an additional hydrogen-bond between repeats , namely from T978 in TM11 . And lastly , towards the cytoplasm , R971 is in close proximity to E414 and N415 , from TM4 , particularly in the T state ( Figure 4 ) . ( D408 is also the acceptor of an additional hydrogen-bond , but within its own repeat , namely S481 from TM6 . ) In the O state , by contrast , all R1-R2 interactions break off , and are replaced by alternative ones within each repeat . D407 rotates to engage the backbone of G403 , one helix-turn above along TM4; K940 retracts towards R1 , and donates hydrogen-bonds to N941 ( TM10 ) and T978; and R971 moves away from E414/N415 , and interacts instead with the backbone carbonyl of L944 and the aromatic ring of F948 , both in TM10 . ( D408 also switches interaction partners , to the backbone of L442 in TM5 , i . e . also within repeat R1 . ) In sum , repeats R1 and R2 appear to exchange between an ‘engaged’ state in the L and T conformations , to a ‘disengaged’ state , in the O conformation . 10 . 7554/eLife . 03145 . 017Figure 4 . Configuration of the proton-dependent conformational switch in the core of the TM domain of AcrB , in the L , T and O states , viewed from the periplasm . The structure corresponds to the asymmetric crystal structure of AcrB at a resolution of 1 . 9 Å ( PDB entry 4DX5 ) . The top panels show a close-up of the proton-binding sites , D407 and D408 , along with neighboring side-chains , including K940 in the foreground and R971 in the background . The lower panels are close-ups of R971 and interacting side-chains in its proximity . The highlighted side-chains are shown along with 2Fo-Fc electron density maps contoured at 1 . 0σ . Note this proton-relay network is precisely at the interface between the two transmembrane repeats , R1 and R2 ( blue and red ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 017 To examine how the local and global changes in the structure of the TM domain of AcrB are related to proton translocation , we carried out two distinct but complementary computational analyses . First , we conducted a series of Monte Carlo simulations of the protonation equilibrium of all aspartate , glutamate and histidine side-chains in the protein under a continuum-electrostatics framework , that is , via the Poisson equation ( ‘Materials and methods’ ) . In these calculations all lysine and arginine side-chains were assumed to be ionized , except for K940 and R971 , which were explicitly analyzed . Figure 5 summarizes the results from these simulations , focusing on the TM domain . Most of the aspartate , glutamate and histidine side-chains were found to be deprotonated across the LTO cycle; many of these aspartate and glutamate are engaged in salt-bridges , particularly on the cytoplasmic side of the protein ( Figure 5A , B ) . A handful of residues , however , change their most probable protonation state in the T to O transition . These include D407 and D408 , in the core of the TM domain , which are ionized in the L and T states but become protonated in the O state ( Figure 5C ) . Interestingly , E346 and D924 , at the periplasmic end of TM2 and TM10 , respectively , are protonated in the L and T states , but become deprotonated in O; conversely , the neighboring H338 ( TM2 ) and D566 ( in the region between TM7 and PC1 subdomain ) are most likely deprotonated in L and T , and become protonated in the O state ( Figure 5C ) . Lastly , the calculations indicate that both K940 and R971 remain protonated throughout the cycle . 10 . 7554/eLife . 03145 . 018Figure 5 . Changes in the computed protonation probability of ionizable residues in the TM domain , across the LTO conformational cycle . The three protomers in the asymmetric crystal structure of AcrB are shown side by side , in a cartoon representation ( white ) , in the same orientation as in Figure 1 . Ionizable residues are represented as van-der-Waals spheres , and colored according to their protonation probability , computed via Monte-Carlo Poisson–Boltzmann calculations ( ‘Materials and methods’ ) . ( A ) Glu , Asp and His residues predicted to be in the deprotonated state in all conformations of the protomer . ( B ) K940 and R971 are predicted to be protonated in the L , T and O states; other lysine and arginine residues are assumed to be protonated in the calculation . ( C ) Glutamate , aspartate and histidine residues whose protonation state is predicted to be inter-dependent with the conformation of the protomer . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 01810 . 7554/eLife . 03145 . 019Figure 5—figure supplement 1 . Electrostatic switch involving K940 and R971 upon protonation of D407 and D408 . The plot shows net electrostatic interactions between K940 , R971 and D407/D408 , in each conformational state of AcrB; ‘other’ indicates the net interaction with all residues in the protein except for K940 , R971 , D407 and D408 . D407 and D408 are deprotonated in states L ( blue ) and T ( yellow ) , and protonated in state O ( red ) . This analysis shows that in the transition from T to O , K940 and R971 lose favorable electrostatic interactions with D407 and D408 , as a result of their becoming protonated; however , by re-orienting and retracting towards their own repeat , that is , R2 ( see Figure 3 ) , they gain much of the lost electrostatic energy , by increasing their favorable interactions with other residues in the protein , by minimizing the repulsion between each other , and by increasing their exposure to solvent . In turn , this reconfiguration results in a lateral displacement of the repeats relative to each other . Electrostatic interaction energies were extracted from a Monte-Carlo Poisson–Boltzmann simulation of the protonation equilibrium of AcrB ( ‘Materials and methods’ ) ; the plot shows average values over 500 , 000 steps of the Monte-Carlo simulation; error bars correspond to the standard-deviation in each case ( not visible in all cases ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 019 As is well known , electrostatic-energy calculations based on the Poisson equation often yield divergent results depending on the value of the protein dielectric constant that is assumed . Although the results just described are generally consistent across the range of most plausible values ( Kukic et al . , 2013 ) , namely 2 to 6 , we sought to confirm our conclusions with a second methodology . In particular we used a series of all-atom molecular dynamics simulations of AcrB protomers embedded in a phospholipid membrane ( Figure 1—figure supplement 2 ) to calculate the protonation free energy of individual sites , relative to side-chain analogs in bulk water ( ‘Materials and methods’ ) . Specifically , we computed the free energy of protonation of D407 , D408 and K940 , in the O state , and of E346 and D924 , in the T conformation . The results , shown in Figure 6 , reveal strong upward shifts in the pKa of D407 ( in the O conformation ) , D408 ( O ) , E346 ( T ) and D924 ( T ) , and a weak downward shift in K940 ( O ) , thus confirming that these side-chains are most likely protonated in those protomer states . As controls we also analyzed E693 ( T ) and K603 ( O ) , which are located on the solvent-exposed surface of the periplasmic PC2 subdomain; as expected , the calculated pKa shifts for these side-chains are small or negligible ( Figure 6 ) . In sum , both all-atom molecular simulations and continuum-electrostatic calculations indicate that in the L and T states , two protons are bound to the TM domain of AcrB at the periplasmic side ( i . e . , D924 and E346 ) , while D407 and D408 , two-thirds of the way across the membrane , are deprotonated; upon transition to the O state , two protons bind to D407 and D408 , while the proton-accepting sites on the periplasmic face become deprotonated . 10 . 7554/eLife . 03145 . 020Figure 6 . Free energy of protonation for selected glutamate , aspartate and lysine residues in the L , T and O states . All values were computed via all-atom molecular simulations in a phospholipid membrane ( ‘Materials and methods’ ) . Computed values for side-chain analogs in solution , whose pKa is known experimentally , provide a means to translate the computed free-energy values into a pKa scale ( dashed lines ) . ( A ) The calculations indicate upward shifts in the pKa of E346 and D924 in the T state , and of D407 and D408 in the O state , relative to acetate in water , whose magnitude indicate that in those conformational states these residues are protonated at physiological pH . By contrast , the pKa of E693 is hardly shifted , consistent with its location on a water-exposed , electrostatically neutral area of the protein surface . ( B ) The pKa of K940 in the O state is also shifted downwards relative to n-butylamine in solution , on account of its being largely unexposed , but does not become deprotonated owing to polar contacts with N941 and T978 ( Figure 4 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 02010 . 7554/eLife . 03145 . 021Figure 6—figure supplement 1 . Statistical analysis of ionic and non-ionic side-chains interactions formed by lysine side-chains in high-resolution crystal structures deposited in the Protein Data Bank . The datasets analyzed comprise crystal structures with non-redundant amino-acid sequences , based on either a 70% or a 30% sequence-identity threshold ( 21 , 780 and 13 , 735 structures , respectively ) . For each lysine side-chain in each structure , we identify the number of polar and/or acidic side-chains with which the lysine side-chain interacts directly , that is , via a direct hydrogen-bond . This analysis shows that the interaction patterns observed for K940 in AcrB ( highlighted in blue/red ) are frequently observed in protein structures; interestingly , the frequency with which two concurrent Glu/Asp interactions are observed , as in the L and T states of AcrB ( blue ) is comparable ( slightly smaller ) to that with which two non-ionic interactions are observed , as in the O state of AcrB ( red ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 021 The proposed changes in the protonation state of D407 and D408 in the transitions from T to O ( protonation ) and O to L ( deprotonation ) are consistent with the ‘disengagement’ and ‘reengagement’ , respectively , of the structural repeats R1 and R2 , as the ionic interactions with K940 would be weakened by protonation of the aspartate side-chains . The finding that K940 remains ionized in the O state might be less intuitive . However , analysis of non-redundant high-resolution structures in the Protein Data Bank reveals thousands of instances in which a lysine side-chain is part of an electrostatic interaction network that does not include any negatively-charged contacts ( Figure 6—figure supplement 1 ) . Moreover , in this O state K940 not only hydrogen-bonds with N941 with T978 ( Figure 4 ) , but also interacts with water , as we will show below . This polar micro-environment explains the small shift in the pKa calculated here . Consistent with this result , measured pKa values for lysine side-chains engineered in a slightly less polar ( and also non-ionic ) environment within the model protein SNase are down-shifted only by ∼2 units ( Damjanović et al . , 2011 ) . As mentioned above , experimental evidence demonstrates that K940 and R971 are indispensable for the proton-driven mechanism of AcrB ( Guan and Nakae , 2001; Murakami and Yamaguchi , 2003; Takatsuka and Nikaido , 2006; Seeger et al . , 2009 ) . Our results suggest that the primary role of these side-chains is not , however , to capture and donate protons; instead , they appear to function as a conformational electrostatic switch coupled to the protonation state of D407 and D408 . In the L and T states both K940 and R971 have strong favorable interactions with ionized D407 and D408 , which exceed their mutual repulsion ( Figure 5—figure supplement 1 ) . Neutralization of D407 and D408 , in the transition to the O state , alters this electrostatic balance . As a result , K940 and R971 reorient to reduce their electrostatic repulsion and to maximize alternative favorable interactions within the protein , particularly within repeat R2 ( Figure 5 , Figure 5—figure supplement 1 ) . We propose that the re-configuration of this reversible electrostatic switch , which is coupled to the protonation and deprotonation of both D407 and D408 , is the trigger for the lateral displacements of repeat R2 relative to R1 observed in the T to O and O to L transitions ( Figure 3; Video 5 , Video 6 ) . To further assess the proposed proton-relay mechanism and its influence on the conformation of the TM domain , we solved the atomic structures of four AcrB variants , namely K940A , R971A , D407N and D408N ( Supplementary file 2 ) , all of which abolish the drug-efflux activity of AcrB ( Guan and Nakae , 2001; Murakami and Yamaguchi , 2003; Takatsuka and Nikaido , 2006; Seeger et al . , 2009 ) . Electron-density maps for these variants could be resolved at resolutions ranging from 2 . 0 to 2 . 3 Å , which are sufficient to discern the precise configuration of side-chains and structural waters inside the TM domain . In all cases , the crystal structures represent an asymmetric configuration of the AcrB trimer . As in the wildtype transporter , this asymmetry originates in the porter domain due to drug binding ( Supplementary file 3A , Supplementary file 3B ) , compounded by the steric coupling between protomers in this region . This asymmetry in the porter domain is important for crystallization , as it facilitates the recognition of designed-ankyrin repeats proteins ( DARPins ) developed to improve wildtype crystals ( Sennhauser et al . , 2007 ) . As in wildtype AcrB , the asymmetry of the porter domain propagates to the transmembrane region , where the protomers also adopt three distinct conformations . None of the substitutions studied caused significant changes in the internal structure of the R1 and R2 repeats , compared to the wildtype L , T and O states ( Supplementary file 3A ) . The relative orientation of these two repeats is also unchanged in the K940A and D408N mutants ( Supplementary file 3C ) , as is the side-chain configuration of the proton-relay network , aside from the mutations ( Figure 7 ) . That is , these two substitutions appear to perturb the conformational cycle of the transporter by altering the energetic balance between the L , T and O states , rather than by precluding the protomers from adopting native-like conformations in each of these states . Indeed , electrostatic calculations analogous to those carried out for the wildtype protein show that in the K940A structure , D408 is constitutively neutralized throughout the cycle . D407 , which is more proximal to R971 , is deprotonated in the L and T states and protonated in the O state , as in the wildtype ( Figure 7 , Figure 7—figure supplement 1 ) . Similarly for the D408N variant , D407 is predicted to become protonated only in the O state , while the substituted N408 is evidently neutral throughout the cycle ( K940 and R971 are protonated in all states in both mutants ) ( Figure 7 ) . These results imply that the ionic interactions across the R1-R2 interface that stabilize the L and T states are weakened in the mutants . By contrast , the O state is probably less affected ( K940A ) or not at all ( D408N ) , since the repeats become disengaged in this state . This change in the energetic balance of the conformational cycle ( which for example would hinder the transition from O to L ) would explain the drastic functional effect of these two mutations . 10 . 7554/eLife . 03145 . 022Figure 7 . Configuration of the proton-binding site in asymmetric crystal structures of four AcrB variants inactive in drug efflux , compared to the wildtype protein . The binding site is viewed and represented as in Figure 4 , omitting the electron-density map . The most probable protonation states of D407 and D408 , based on Poisson–Boltzmann Monte-Carlo simulations ( wildtype and mutants ) as well as all-atom free-energy perturbation molecular dynamics simulations ( wildtype ) , are indicated in each case with ‘H+’ . K940 and R971 are protonated in all cases , according to these calculations . Hydrogen-bonds identified on the basis of the distance and orientation of potential donors and acceptors , are indicated with dashed lines . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 02210 . 7554/eLife . 03145 . 023Figure 7—figure supplement 1 . Protonation probability ( % ) of selected side-chains in the transmembrane domain of AcrB , in crystal structures of wildtype and mutagenized variants of the transporter . Each of the protonation probability values shown is an average of the results of 10 independent Monte-Carlo/Poisson–Boltzmann simulations of the coupled protonation equilibrium of all glutamate , aspartate and histidine side-chains , plus K940 and R971 , in full-length AcrB trimers ( see ‘Materials and methods’ for further details ) . Standard deviations are also shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 023 Electrostatic calculations for the R971A and D407N crystal structures indicate that , like K940A and D408N , these variants lack the ability to bind and release two protons ( Figure 7—figure supplement 1 ) . In R971A , like in K940A , a first proton is already bound to the protomer in the L and T states ( alternating between D407 and D408 , respectively ) and a second proton binds in the O state ( Figure 7 ) . In D407N , like in D408N , only one proton is loaded ( on D408 ) , and only in the O state ( Figure 7 ) . Thus , also in these two variants is the key ionic interaction network at the R1-R2 interface partially neutralized in the L and T states . The R971A and D407N substitutions , however , not only perturb the energetics of the conformational cycle and the transduction of the proton electrochemical gradient , but also seem to preclude the protomers from adopting all the native-like conformations , particularly the T state ( Figure 7 ) . The structure of R971A shows that K940 is engaged only with D408 in the L state ( while D407 is protonated ) . In the T state , K940 switches towards D407 ( while D408 is protonated ) , adopting a configuration that is arguably an intermediate between the native T and O states: first , in that K940 retracts away from the repeat interface , unhindered by the now absent repulsion with R971 , while still paired with D407; and second , in that as a result D407 loses its hydrogen bond to T978 across the R1-R2 interface . Consistent with our hypothesis that the structural configuration of the proton-relay network is intimately coupled to the displacements of the 5-helix repeats , the arrangement of these repeats in the T protomer of the R971A crystal structure is an intermediate between the T and O states of the wildtype structure ( Figure 8 ) ; in fact , this altered conformation is more similar to the latter ( RMSD values of 2 . 4 vs 1 . 6 Å , respectively—Supplementary file 3C ) . 10 . 7554/eLife . 03145 . 024Figure 8 . Relative motions of transmembrane repeats R1 and R2 in wildtype AcrB and in the R971A and D407N variants , in the T to O transition . For clarity , only the three transmembrane helices at the interface between repeats are shown , namely TM4-TM6 ( R1 , blue cartoons ) and TM10-TM12 ( R2 , red cartoons ) , respectively . The structural superimpositions shown are optimal overlays of TM4-TM6 , and thus highlight the differences in the orientation of TM10-TM12 relative to TM4-TM6 . This comparison shows that the T state in the crystal structures of the R971A and D407N mutants is an intermediate between the T and O conformations in wildtype AcrB . The conformation of the O state , however , is largely unaffected by these mutations . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 024 The effect of the D407N substitution is subtler than that of R971A , but also underscores the relationship between the local configuration of the proton-relay network and the organization of the transmembrane repeats . The D407N structure shows that although the substituted asparagine can accept a hydrogen-bond from K940 in the L and T states , its orientation is geometrically inconsistent with the second hydrogen-bond with T978 seen in the wildtype ( Figure 7 ) . In the L state this appears to be compensated by an alternative hydrogen-bond donated by N941 , but this interaction is not feasible in the T state . In addition , the asparagine substitution removes the attractive electrostatic interaction between D407 with R971 . In sum , the interaction network at the R1-R2 interface is partially disengaged . Consequently , the T state in the D407N structure shows R1 and R2 partially displaced , in a conformation that is again intermediate between the wildtype T and O states ( Figure 8; Supplementary file 3C ) . As mentioned above , the crystallization of these variant structures was facilitated by DARPin ligands , which recognize the porter domain of AcrB in its asymmetric form ( Sennhauser et al . , 2007; Eicher et al . , 2012 ) . Because the porter and transmembrane domains are necessarily coupled , it is conceivable that the DARPins inhibit the structural impact of the mutations , that is , only the strongest perturbations ( R971A and D407N ) would translate into noticeable structural differences in our experiment . Thus , we cannot rule out that the lack of activity of these mutants reflects structural perturbations more significant than those observed here . Nonetheless , even the relatively modest changes revealed by the structures presented here support the conclusions of our analysis of wildtype AcrB . That is , that there is a causal relationship between the displacements of the repeats R1 and R2 within the TM domain and the configuration of the proton-dependent electrostatic network at the R1-R2 interface; that there is a conformational and electrostatic coupling between R971 and K940 and the two aspartate side-chains that serve as proton-binding sites; and lastly , that the transitions from T to O and O to L , which power the peristaltic pump mechanism of the porter domain , entail binding and release of two protons at D407 and D408 . Proton translocation across membrane proteins necessarily requires a pathway of water molecules to facilitate access from the surrounding solution , and to mediate proton hopping between protonatable side-chains . In the case of proton-driven secondary transporters , these pathways must also be dependent on the conformational state of the protein , that is , they must be consistent with an alternating-access mechanism , so as to preclude leakage and ensure strict coupling of substrate and proton transport . To examine whether the conformational cycle of AcrB indeed implies changes in proton accessibility , we carried out an all-atom molecular dynamics simulation of the complete trimer in a lipid membrane , and analyzed the formation and persistence of hydration channels across the TM domain . The results , summarized in Figure 9A , indicate that the LTO conformational cycle described above does indeed involve a mechanism of alternating access . More specifically , we observe that the L state permits the formation of two persistent water channels open to the cytoplasm , at the interface between repeats R1 and R2 , and reaching into the proton-binding site; access from the periplasmic space is , however , closed ( Figure 9A ) . In the T state , by contrast , two narrow water wires form on the periplasmic face of the TM domain , again reaching into the proton-binding site , and approximately along the interface between the two repeats R1 and R2 ( Figure 9A ) . Interestingly , E346 and D924 flank the entrance of each of these water wires; as mentioned earlier , both these side-chains are likely to be protonated in the L and T states and deprotonated in the O state ( Figure 4 , Figure 5 ) , which suggests that E346 and D924 facilitate proton uptake by providing transient binding sites en route to D407/D408 ( Fischer and Kandt , 2011 ) . On the cytoplasmic side , we also observed a narrow water wire , flanked by R971 , which is thus unlikely to sustain proton transfer , as discussed below . Finally , the O state features two water channels into the proton-binding site that are highly similar to those seen in the L conformation , that is , open to the cytoplasm , while access from the periplasmic space is also occluded ( Figure 9A ) . 10 . 7554/eLife . 03145 . 025Figure 9 . Alternating access to the proton-binding site in AcrB . ( A ) Conformation-dependent water channels within the TM domain of AcrB , observed in all-atom simulations in a phospholipid membrane . Transmembrane helices are represented as cartoons and colored as in Figure 1; for clarity , TM7 , TM9 and TM12 in repeat R2 are omitted . The locations of D407 , D408 , K940 , R971 , E346 and D924 are indicated with green spheres representing their Cα atoms . The water channels are represented by average density maps computed from the molecular dynamics trajectory , and depicted as an iso-density surface ( gray ) . Based on this analysis , the proton-binding site is open to the cytoplasm in the L and O states , via two possible channels ( solid black lines ) , whereas in the T state it is open to the periplasm , also via two water wires ( solid black lines ) . A third narrow wire in the T state seems to connect the proton-binding site to the cytoplasm ( dashed black lines ) , but the positively charged R971 , which traverses this water wire , likely blocks H+ leakage . ( B and C ) Crystallographic water molecules ( gray spheres ) detected within the TM domain of AcrB , in each of the protomer states of the wildtype structure , as well as in the T protomer of the D407N and R971A variants . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 025 A striking feature of these putative access pathways for protons is that they are , for the most part , highly dynamic and permit individual water molecules to frequently exchange between the bulk solution and the close vicinity of the proton-binding site ( Figure 10A ) . For the L state , for example , we observed more than 900 exchange events in a 250-ns period; about 70% occur via the cytoplasmic channel formed between helices TM5 ( repeat R1 ) and TM10 ( repeat R2 ) , while the remainder proceeds via the channel formed between TM4 ( R1 ) and TM11 ( R2 ) . Likewise for the O state , with over 600 exchange events in the same time-period , distributed over the two channels , roughly in a 2:1 ratio . In the T state , the periplasmic pathway flanked by TM2-TM4 and TM11 seems to be the most dynamic , although much less so than the cytoplasmic channels in L and O ( we observed about 30 exchange events ) , while the periplasmic wire flanked by TM10 consists of a series of water molecules that are static on the simulation time-scale . 10 . 7554/eLife . 03145 . 026Figure 10 . Dynamic exchange of water molecules between the proton-binding site in the core of the TM domain of AcrB and the bulk solution at either the periplasmic or cytoplasmic sides of the membrane and proposed role of R971 . ( A ) A random selection of single-water trajectories extracted from the molecular dynamics simulation are shown as blue lines , cut-off at 30 Å from the proton-binding site . The location of residues D407 , D408 , K940 and R971 is indicated by their Cα atoms , shown as green spheres . Transmembrane helices in each protomer/state are represented as cartoons ( white ) ; for clarity , TM7 , TM9 and TM12 in repeat R2 are omitted . ( B ) Proposed role of R971 as a selectivity filter against cytoplasmic H+ leakage in the T state of AcrB . The plot quantifies the frequency of water exchanges between the proton-binding site within the TM domain of AcrB and the bulk solution at the cytoplasmic side of the membrane , as a function of the proximity of the individual molecular trajectories to R971 . In the T state , almost 100% of the water trajectories approach R971 within 10 Å , and therefore this water wire ( dashed lines in Figure 9 ) is highly unlikely to sustain H+ conduction , for electrostatic reasons . In the L and O states , by contrast , 40% of the exchanging water molecules follow trajectories that do not approach R971 ( solid lines in Figure 9 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 026 As mentioned , in the T state we also observed a significant number of water molecules exchanging between the proton-binding site and the cytoplasm . However , the trajectories followed by these water molecules invariably enter or exit the TM domain in close proximity to R971 ( within 10 Å ) ( Figure 10B ) . This positively charged side-chain very likely imposes a very large energetic barrier for proton diffusion to and from the cytoplasm , despite the presence of water . Such an electrostatic barrier would be analogous , for example , to that observed in the selectivity filter of aquaporins , which is also lined by an arginine side-chain ( Kosinska Eriksson et al . , 2013 ) . By contrast , almost half of the water-permeation events in the L and O states involve molecular trajectories that are distant from R971 at all times ( Figure 10B ) . Likewise , no large electrostatic barriers for protons are apparent along the periplasmic pathways in the T state . That the hydration of the TM domain is conformationally dependent is entirely consistent with the electron densities from ordered water molecules in the X-ray structures of wildtype and variant AcrB . As shown in Figure 9B , the structure of the T state in wildtype AcrB features the abovementioned continuous water wire flanked by TM10 , connecting the periplasmic space and D407/D408 , with D924 at its entrance . The second outward-open wire , with E346 at its entrance and running along the interface between TM2-TM4 and TM11 , cannot be discerned in the wildtype structure , but interestingly , is partially resolved in the structures of D407N and R971A ( Figure 9C ) . As discussed earlier , in these crystals the T protomer is captured in an intermediate conformation between wildtype T and O , in which the lateral displacement of repeat R2 is already underway ( Figure 8 ) . In the native membrane and at room temperature , the two repeats would be much more dynamic than what appears in the crystal lattice , and thus we envisage that both water wires co-exist , also in the wildtype protein , as we observe in the simulation . In any case , it is apparent that no water wires connect the central proton-binding site and the periplasmic space in the L and O states , in any of the structures . By contrast , a significant number of ordered water molecules are observed in the intracellular side of these protomers . According to our simulation , the cytoplasmic channels , and particularly that between TM5 and TM10 , are highly dynamic , and thus electron densities in this region would be poorly defined . Nevertheless , ordered water can be clearly discerned in the proximity of the proton-binding site , particularly in the L state ( Figure 9B ) . In sum , our data indicate that throughout the conformational cycle of AcrB the proton-binding sites within the TM domain become alternately accessible to the periplasm or the cytoplasm . As mentioned , this is a necessary condition in order for the transporter to harness the proton-motive-force . Interestingly , two distinct types of relative motion of repeats R1 and R2 result in alternating access , namely a rocking motion during the L to T transition , with no protons bound , and a shear motion in the T to O transition , in the protonated form . This finding indicates that other transporters may also follow significantly different conformational pathways in the substrate-bound and apo segments of their functional cycle , contrary to what is often assumed . To our knowledge , however , AcrB is the only secondary-transporter for which two distinct structural transitions resulting in alternating access have been described to date . As described above , AcrB consists of well-defined structural elements in both its periplasmic and membrane domains , whose relative arrangement changes to a significant degree throughout the conformational cycle of the transporter . In the periplasmic domain , these rearrangements reflect substrate loading from the periplasm , the re-shaping of the hydrophobic binding site , and its release into the TolC lumen . In the membrane domain , water pathways across the protein become reconfigured to facilitate access from either side of the membrane , and a network of ionic and H-bonding interactions is re-organized as a result of protonation and deprotonation events . Taken together , these observations reveal an unprecedented antiport strategy that involves the remote coupling of two alternating-access transport mechanisms , that is , one for drugs and another for protons , within each of the AcrB protomers , which we summarize in Figure 11 . In the L to T transition , substrate binding in the deep binding pocket of the porter domain causes a structural change within the PN2/PC1 unit . This change translates into the downward displacement of TM2 , which pushes on Iα and causes it to swing away , along with whole of the R2 repeat ( Figure 3 ) . In this transition , the interaction network formed by the proton-relay triad , D407 , D408 and K940 , remains engaged ( Figure 4 ) , with all three side-chains ionized ( Figure 5 , Figure 6 ) , and seems to provide the hinge point of the swinging motion of R1 and R2 relative to each other ( Figure 3 ) . Importantly , this motion leads to alternating access of pathways for protons from either side of the membrane . Specifically , while in the L state the triad is accessible only from the cytoplasm , in the T state protons can only enter the TM domain from the periplasm ( Figure 9 , Figure 10 ) . In both cases , proton transfer is highly likely to be mediated by water channels formed at the interface between repeats , which open or close as the repeats themselves rearrange relative to each other . 10 . 7554/eLife . 03145 . 027Figure 11 . Proposed mechanism of drug-proton antiport in AcrB . ( A ) The scheme shows the conformational cycle of one of the protomers; the porter domain is viewed from the outer membrane , and the TM domain is viewed along the membrane plane . The structural repeats in the TM and porter domains , R1 and R2 , and PC1/PN2 and PN1/PC2 , respectively , are colored as in Figure 1 . Likewise for the TM2 and TM8 , which flank R1 and R2 and couple them to the repeats in the porter domain . The conformational state ( L/T/O ) of each protomer is indicated alongside the proposed location of the substrate ( D ) and the protons ( H+ ) in each state . Residues in the proton relay network formed by K940 , R971 , D407 and D408 , which controls the relative orientation of R1 and R2 , are indicated with ‘+’ and ‘−’ symbols . The series of states proposed for the TM domain is based on the analysis of protonation states and water accessibility of wildtype AcrB and its variants , reported above . The model postulates that TM2 and TM8 mediate the strict coupling between these two conformational cycles . ( B ) The scheme shows the conformational cycle of the porter-domain trimer , viewed from the outer-membrane . The specific series of states proposed for the porter domain derives from analysis of potential steric clashes at the protomer–protomer interface in all possible combinations of L , T and O conformations ( Figure 11—figure supplement 1 ) . Note that the frequency of protomers in the O state is minimized , relative to other possible cycles , while that of protomers in the L and T state is maximized; we propose this kinetically advantageous , in that it would result in minimum back-flow of substrates from the exit pore , and maximal capture from the periplasmic space . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 02710 . 7554/eLife . 03145 . 028Figure 11—figure supplement 1 . Mechanistic significance of the conformational coupling among protomers . ( A ) Steric clashes in hypothetical models of the porter domain of AcrB , in all possible non-symmetric permutations of the L , T and O conformations . The steric clash in a given trimer configuration is defined as the volume of the overlapping atoms from different protomers at the three interfaces . The trimer models were generated via rigid-body rotations of the PC1/PN2 and PN1/PC2 sub-domains around the trimer axis , and optimized with a side-chain prediction algorithm ( ‘Materials and methods’ ) . The combinations shown in green are the most feasible from a steric standpoint; these are also sufficient to draw a complete transport cycle that , arguably , would be also ideal from a kinetic standpoint , as it would minimize the chances of drug re-binding after extrusion into the TolC lumen ( see manuscript ) . The conformations shown in red are likely to be energetically prohibited , because LO and OO interfaces would require extensive deformations in the protomers structure . ( B ) For alternative hypothetical transport cycles of the AcrB trimer , ratio of the number of instances in which a protomer adopts either the L or T state , relative to the O state . Only non-symmetric trimer states are considered . The conformational cycle of an uncoupled trimer features the largest number of possible trimer configurations , but also the largest proportion of O states . In the coupled trimer , many of those trimer configurations are excluded because speficic protomer–protomer interfaces are sterically incompatible , for example , OO and LO ( A ) . The plot shows that these specific exclusions imply an optimization of the LT/O ratio in the conformational cycle , which is likely to translate into a more kinetically efficient efflux system . DOI: http://dx . doi . org/10 . 7554/eLife . 03145 . 028 In the T to O transition , D407 , D408 and K940 break away from each other ( Figure 4 ) , following binding of one proton to each of the aspartate side-chains ( Figure 5 , Figure 6 ) . K940 remains in the ionized , protonated state , and retracts towards TM10/TM11 to compensate for the loss of interaction partners . R971 ( on TM11 ) also reconfigures in response to these events , rotating towards TM10 . As a result , the repeats R1 and R2 become disengaged , and R2 ( along with Iα ) moves laterally relative to R1 ( Figure 3 ) . This displacement allows TM2 to return to the up conformation , and importantly , enables TM8 to come into close proximity to R2 . The displacement of TM8 causes the PN1/PC2 repeat in the porter domain to move , as a rigid body , against PN2/PC1 , thus closing the binding cleft to the periplasmic space , and triggers instead the opening of an exit pathway towards the funnel domain . The ability of TM8 to steer the motion of PN1/PC2 in the T to O step ( Video 5 ) seems to be facilitated by the elongation of the helical fold of TM8 , on the periplasmic side , upon transition from L to T ( Video 4 ) ; this naturally facilitates its coupling to the motion of PN1/PC2 . Crucially , in the O state the proton-relay site is again accessible to the cytoplasm , and not to the periplasm ( Figure 9 , Figure 10 ) . Thus , deprotonation will imply a release of the bound protons down the electrochemical gradient . Proton release into the cell interior also causes the R1 and R2 repeats to re-engage ( Figure 4 ) , and thus the dissociation of TM8 from R2 when reforming the L state ( Figure 3 ) . This enables the re-opening of the drug-binding cleft between PC1 and PC2 , exposing the access binding pocket ( Nakashima et al . , 2011; Eicher et al . , 2012 ) to the periplasm , ready to sequester a new drug molecule . It is worth noting that the directionality of this mechanism is entirely encoded structurally , for each of the protomers . Proton uptake leads to substrate export ( instead of symport ) for two reasons: first , both proton- and substrate-binding sites are concurrently open to the periplasm in the T state , while in the O state they become open to the cytoplasm and the TolC lumen , respectively; second , both porter and membrane domains are able to re-open to the periplasm after releasing their cargo , that is , in their apo state ( Figure 11A , drug access pocket and proton loading ) . Thus , the free-energy gain associated with downhill proton permeation compensates the cost of uphill efflux of cytotoxic compounds . This energy-transduction process is reminiscent of the binding-change mechanism of the F1Fo-ATP synthase , which also utilizes the energy harnessed from the transmembrane electrochemical proton gradient to release synthesized ATP molecules from their binding sites in the α/β subunits ( Boyer , 1997; Pos , 2009 ) . Although the mechanism and energetics of transport in AcrB can be fully rationalized in terms of the individual protomers , the extensive trimer interface within the porter domain suggests that the protomers are conformationally coupled . Indeed , transport of certain cephalosporins and penicillins has been shown to be highly cooperative ( Nagano and Nikaido , 2009; Lim and Nikaido , 2010 ) . How the cross-talk among protomers might benefit the antiport mechanism is , however , not immediately evident . From an architectural standpoint , trimerization is necessary for AcrB to form a pore leading into the TolC channel , but why would the protomers not cycle independently from each other ? A disadvantage of independent protomer cycling would be that a drug released into the exit pore by a protomer in the O state would often be exposed to a second protomer also in the O state , which would enhance the probability of re-binding . Such event would not likely result in drug re-uptake ( which would require uphill proton transport ) but efflux would be surely hindered from a kinetic standpoint , because the drug concentration within TolC is probably very large . Ideally , therefore , the number of trimer configurations in which one or more protomer are in the O state ought to be somehow minimized . Following the same kinetic argument , it would be advantageous if AcrB maximizes the number of trimer configurations featuring the L and T states in one or more protomers , so as to accelerate capture ( or re-capture ) of the substrate from the periplasmic space or the inner membrane . Remarkably , analysis of computer-generated models of the porter domain in all non-symmetric permutations of the L , T and O states ( ‘Materials and methods’ ) indicates that this kind of kinetic optimization is what emerges from the conformational coupling among protomers . A quantification of the steric clashes between protomers in these hypothetical models indicates that OO and LO interfaces ( counter-clockwise , viewed from TolC ) are highly incompatible , and that OT is also suboptimal . Thus , most of the combinations that include one or more O states in the trimer would be energetically unfeasible ( Figure 11—figure supplement 1 ) . Figure 11 depicts the conformational cycle of the porter domain that involves only the most plausible combinations , alongside the cycle of the TM domain described earlier . Throughout this cycle , the proportion of protomers in either the L or T state relative to those in the O state is fourfold greater than in a hypothetical uncoupled trimer ( Figure 11—figure supplement 1 ) . That is , the AcrB protomers are substantially more efficient efflux pumps if they are coupled; not because the protomers energize each other , but because collectively they are able to restrict their conformational pathways so as to achieve the most productive mechanism . New high-resolution crystal structures of wildtype and AcrB variants , systematically analyzed through diverse computational methods , have enabled us to formulate a novel , clear-cut theory of the mechanism of proton-coupled drug-efflux in this very complex transport system . On the basis of this theory , which is consistent with existing biochemical , structural and functional data , additional and more precise experiments and computations may now be designed to further establish the proposed molecular mechanism of transport and energy transduction . It will also be of interest to extrapolate these findings to other homologues in the RND superfamily . Such investigations could potentially aid the development of novel pharmacological approaches , such as conformational inhibitors , against Gram-negative multidrug resistant pathogens .
E . coli C43 ( DE3 ) ( Miroux and Walker , 1996 ) harboring mutant derivatives of pET24acrBHis ( Pos and Diederichs , 2002; Seeger et al . , 2009 ) were used for protein overexpression . LB medium and LB agar ( Sambrook et al . , 1989 ) were used for routine bacterial growth at 37°C . Terrific broth ( Tartoff and Hobbs , 1987 ) was used for protein overexpression . Kanamycin ( Applichem , Germany ) was applied at 50 μg ml−1 ( Kan50 ) . Overexpression and membrane preparation of wildtype and mutant AcrB was carried out as described previously ( Pos and Diederichs , 2002; Seeger et al . , 2006 ) . DARPin clone 1108_19 ( Sennhauser et al . , 2007 ) was overexpressed and purified according to Binz et al . ( 2003 ) . To crystallize the AcrB/DARPin complex , the method described by Sennhauser et al . ( 2007 ) with n-dodecyl-β-D-maltoside as detergent ( Glycon Biochemicals , Germany ) was applied . For all AcrB variants except R971A , minocycline was added prior to crystallization at a concentration of 2 mM ( Sigma-Aldrich , Germany ) . All crystals were grown by the hanging drop vapor diffusion method at 0 . 05 M ADA buffer pH 6 . 5 , 7–9% polyethylene glycol ( PEG ) 4000 , 6–10% glycerol , 0 . 2 M ( NH4 ) SO4 . Cryprotection was achieved by transfer of the crystals to the same buffer containing 30% glycerol and flash freezing into liquid N2 . Datasets of P212121 crystals were collected at beamline X06SA ( wavelength 0 . 8–1 . 0 Å ) of the Swiss Light Source ( Paul Scherrer Institut , Villigen , Switzerland ) . Data reduction was done with the XDS package ( Kabsch , 2010 ) . The structures were solved by molecular replacement using MOLREP ( Vagin and Teplyakov , 2010 ) or PHASER ( McCoy et al . , 2007 ) . As search model pdb entry 4DX5 ( Eicher et al . , 2012 ) was used and model rebuilding was employed using COOT ( Emsley et al . , 2010 ) . Refinement was carried out with PHENIX ( Adams et al . , 2010 ) and REFMAC5 ( Murshudov et al . , 2011 ) and additional water molecules appended . Model rebuilding and water molecule analysis was done employing COOT ( Emsley et al . , 2010 ) . Figures were created using PyMOL ( The PyMOL Molecular Graphics System , Version 1 . 5 . 0 . 3 Schrödinger LLC , Oregon , USA ) . Protonation probabilities were calculated using two independent methodologies . Wildtype and mutagenized AcrB structures were analyzed via Monte-Carlo/Poisson-equation simulations of the global protonation equilibrium in the complete trimer . In addition , selected ionizable side-chains in the wildtype transporter were analyzed via all-atom molecular dynamics simulations of individual protomers , using the free-energy perturbation ( FEP ) method . To assess the most likely protonation state of the ionizable side-chains in AcrB , we carried out a series of Metropolis Monte-Carlo simulations of the global protonation equilibrium of the protein at fixed pH . In a first set of calculations , we analyzed in each protomer separately , including all ionizable side-chains in the calculation . All lysine and arginine side-chains were consistently predicted to be protonated at the pH and dielectric constant values used in these calculations ( see below ) . Thus , in subsequent simulations , in which the complete trimer was considered , all lysine and arginine side-chains were fixed in their ionized state , except for K940 and R971 , which were explicitly re-analyzed along with all aspartate , histidine and glutamate residues ( 306 side-chains in total ) . Each Monte Carlo simulation was initiated with a random configuration of protonation states , and consists of five cycles of temperature annealing and a subsequent equilibration . In the annealing phases , the temperature was raised to 400 K and then gradually reduced to 300 K over 100 , 000 simulation steps . The equilibration phases , in which the temperature was kept at 300 K , lasted 500 , 000 steps . The protonation probability of each group was determined by averaging the ionisation states sampled during the equilibration phase of the final cycle of the simulation . This procedure was repeated 10 times with independent starting configurations , resulting in a global average; the typical statistical error was <2% . To evaluate the energy changes associated with the Monte Carlo moves we used a continuum electrostatic model , that is , the crystal structure of the AcrB trimer is surrounded by a high-dielectric medium representing water , except for a low-dielectric rectangular slab surrounding the transmembrane domain , which mimics the lipid membrane . Specifically , we used the linearized Poisson-Boltzmann equation solver implemented in CHARMM c34a2 ( Brooks et al . , 2009 ) to set up the continuum model and to compute all the necessary electrostatic energy contributions . In all simulations the dielectric constant of the solvent was 80 , and that of the membrane was 2 . The results described in the manuscript and represented in Figure 5 and Figure 6 correspond to a protein dielectric constant of 4; alternative values ranging from 2 to 6 were also explored , which resulted in analogous conclusions ( not shown ) . The atomic charges employed are those in the CHARMM27 force field ( MacKerell et al . , 1998 ) . The atomic radii are those previously optimized for continuum electrostatic calculations based on the CHARMM27 force field ( Nina et al . , 1997 ) . Non-standard radii were adapted according to chemical similarity , for example , the radius of the Nζ atom in the neutral lysine was considered to be equal to that of the side-chain nitrogen atom of asparagine/glutamine . The reference aqueous-phase pKa values were: aspartate , 4 . 0; glutamate , 4 . 4 , histidine , 6 . 3; lysine , 10 . 4; and arginine , 12 . 0 . Residues on the periplasmic side of the protein were assumed to exchange protons with a solution at pHout = 5 . 5 , while those on the cytoplasmic side exchanged with a solution at pHin = 7 . 5; interpolated pH values in this range were used for residues across the membrane . Calculations with an alternative value of pHout = pHin = 7 . 5 led to analogous conclusions ( not shown ) . All molecular dynamics simulations were carried out at 298 K and 1 atm . Three independent all-atom systems were studied , namely the complete asymmetric AcrB trimer and truncated constructs of the T and O protomers comprising the transmembrane and porter domains only ( Figure 1—figure supplement 2 ) . In all cases the protein structures were embedded in a hydrated POPC bilayer using GRIFFIN ( Staritzbichler et al . , 2011 ) . All simulations were initiated with the high-resolution ( 1 . 9 Å ) crystal structure of wildtype AcrB ( PDB entry 4DX5 ) . The simulation system for the complete AcrB trimer includes ∼369 , 000 atoms , enclosed in a periodic orthorhombic box of dimensions ∼150 × 150 × 160 Å . This system was simulated for 250 ns ( after equilibration ) to assess the formation and dynamics of water channels within the transmembrane domain . The protomer systems include ∼101 , 000 atoms in a periodic orthorhombic box of dimensions ∼90 × 90 × 120 Å . These systems were used to evaluate the most likely protonation state of selected side-chains using the FEP method . Each of these calculations entailed a total of ∼80 ns of simulation ( after equilibration ) . Three independent all-atom simulation systems were studied , namely the complete asymmetric AcrB trimer ( residues 1 to 1044 in each protomer ) ( Figure 1—figure supplement 2 ) ; a truncated constructs of the T protomer , comprising the transmembrane and porter domains only ( residues 1 to 182 , 275 to 723 and 813 to 1044 ) ( Figure 1—figure supplement 2 ) ; and an analogous truncated construct of the O protomer . In all cases the protein structures were embedded in a hydrated phospholipid ( POPC ) bilayer using the GRIFFIN method ( Staritzbichler et al . , 2011 ) , adding Na+ counter ions when necessary to neutralize the total charge of the system . This system was employed to assess the formation and dynamics of water channels within the transmembrane domain . A minocycline molecule was bound to the porter domain in the T protomer . D407 and D408 were deprotonated in the L and T protomers , and protonated in the O protomer; K940 and R971 were protonated in all protomers . The simulation was initiated with the high-resolution ( 1 . 9 Å ) crystal structure of wildtype AcrB ( PDB entry 4DX5 ) , preserving crystallographic water molecules . Additional water molecules within the protein structure were placed with DOWSER ( http://danger . med . unc . edu/hermans/dowser/dowser . htm ) . To optimize the protein-solvent and protein-lipid interfaces in the model , and to thermalize the system , a series of short simulations ( 10 ns ) were initially carried out with gradually weaker positional restraints applied to the protein and the crystallographic and DOWSER-added water molecules ( excluding hydrogen atoms ) . To enable the protein to tumble , the equilibration phase was extended ( 5 ns ) after replacing the positional restraints with collective conformational restraints ( RMSD ) , applied first to the whole protein , and subsequently to each of three transmembrane domains separately . In the last state of the equilibration ( 10 ns ) , only the configuration of the proton-relay network was preserved via inter-residue distance restraints ( flat-bottom harmonic potential ) . Finally , a fully unrestrained simulation was carried out for 250 ns . The truncated-protomer systems were used to re-evaluate the most likely protonation state of selected side-chains using the free-energy perturbation ( FEP ) method; the substantial computational cost of this methodology is what motivated the truncation of the protein . The preparation and equilibration of these reduced simulation systems was analogous to the procedure followed for the trimer . In the final production phase ( 90 ns ) , however , only the transmembrane domain was fully unrestrained . The conformation of the porter domain was preserved using a harmonic restraint on its RMSD relative to the crystal structure , to preclude any distortions resulting from the artefactual truncation of the funnel domain . Importantly , the force constant of this harmonic restraint was selected so that the structural fluctuations of the porter domain in the truncated-protomer system are comparable to those observed in the unrestrained simulations of the complete trimer ( not shown ) . Thus , the dynamics of the transmembrane domain during the free-energy calculations is unlikely to be significantly affected by the truncation . For both T and O protomers , snapshots after 90 ns of simulation were extracted and used to initiate the FEP calculations . Here , the protonation state of a given side-chain is exchanged alchemically , using a step-wise protocol controlled by a parameter λ that reflects the weight of either protonation state in the potential energy function of the system . All FEP calculations were carried out in the forward and backward directions , with 38 intermediate λ steps ( 33 for D408 in the O state ) . The simulation time per λ step was 1 ns; the initial 100 ps of each simulation were considered as equilibration , and so free-energy increments were obtained from the final 900 ps . All molecular dynamics simulations were carried out with NAMD 2 . 7 ( Phillips et al . , 2005 ) , using an integration time-step of 2 fs , periodic boundary conditions , and a Langevin thermostat set to 298 K . The pressure along the direction perpendicular to the membrane was maintained at 1 atm using a Nose-Hoover Langevin piston , while keeping the surface area of the membrane constant ( ∼69 Å2 per lipid ) . Electrostatic interactions were calculated using the Particle-Mesh-Ewald algorithm , with a real-space cut-off of 12 Å . A shifted Lennard-Jones potential , also cut-off at 12 Å , was used to compute van-der-Waals interactions . The CHARMM27/CMAP force field for proteins and lipids ( MacKerell et al . , 1998 , 2004 ) was used in all calculations . Force field parameters for minocycline were taken from Aleksandrov and Simonson ( 2009 ) . Water density maps were calculated with VMD ( Humphrey et al . , 1996 ) . The frequency of hydrogen-bonded interaction networks involving lysine side-chains and other polar and/or acidic residues was quantified using a subset of entries in the Protein Data Bank that consists of all protein X-ray structures of resolution 3 . 0 Å or better , excluding redundant entries with sequence identity greater than either 30% or 70% . A distance threshold of 3 . 0 Å was used to identify hydrogen-bonds donated by lysine Nζ atoms to potential acceptor oxygen atoms in either Asp ( Oδ ) , Asn ( Oδ ) , Glu ( Oε ) , Gln ( Oε ) , Ser ( Oγ ) or Thr ( Oγ ) side-chains . Alternative structures of the porter domain were modeled in all possible permutations of the L , T and O conformations , via rigid-body rotations of the PC1/PN2 and PN1/PC2 sub-domains around the trimer axis . All side-chains in all models ( including the crystallographic LTO state ) were then re-built using SCWRL4 ( Krivov et al . , 2009 ) . The solvent-excluded surface ( probe radius of 1 . 4 Å ) of each protomer in each model was then calculated with MSMS ( Sanner et al . , 1996 ) , and mapped onto a fine cubic grid . The overlap volume at each of the three promoter interfaces was computed by counting the number of grid-points concurrently inside two opposing surfaces . The models were ranked according to the total value of the overlapping volume .
Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria , and are essential for cholesterol metabolism and embryonic development in humans . We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB , the active component of the major efflux system AcrAB-TolC in Escherichia coli , and one most complex and intriguing membrane transporters known to date . Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer , namely one for protons in the transmembrane region and another for drugs in the periplasmic domain , 50 Å apart . Each of these cycles entails two distinct types of collective motions of two structural repeats , coupled by flanking α-helices that project from the membrane . Moreover , we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system .
The interior of living cells is separated from their external environment by an enveloping membrane that serves as a protective barrier . To regulate the chemical composition of their interior , cells are equipped with specialized proteins in their membranes that move substances in and out of cells . Membrane proteins that expel molecules from the inside to the outside of the cell are called efflux pumps . In Escherichia coli bacteria , an efflux pump known as AcrB is part of a system that removes toxic substances from the bacterial cell—such as the antibiotics used to treat bacterial infections . AcrB and other closely related efflux pumps in pathogenic bacteria are often polyspecific transporters—they can transport a large number of different toxic molecules . These efflux pump systems are also more active in bacteria that have been targeted by antibiotics , and therefore they help bacteria to evolve resistance to multiple drugs . The emergence of bacterial multi-drug resistance is a global threat to human health; to combat this phenomenon , it is essential to understand its molecular basis . Each AcrB protein has three main parts or domains . The periplasmic domain , which is located between the two membranes that surround E . coli , works via an ‘alternating-access cycle’; that is , the shape of the periplasmic domain changes between three different forms in such a way that antibiotic molecules are first captured and subsequently squeezed through the protein towards the outside of the cell . However , the mechanism of the transmembrane domain—which is embedded in the innermost membrane of the bacterium and is the source of energy for the transport process—was not understood . Here , Eicher et al . use X-ray crystallography to examine the three-dimensional structures of the AcrB efflux pump—and several inactive variants—in high detail . Combining these results with computer simulations reveals the mechanism used by the transmembrane domain to take up protons from the exterior and transport them into the cell . Proton transport also proceeds according to an alternating-access mechanism—and , although the transmembrane and periplasmic domains are far apart , their movements are tightly linked . Thus , because proton uptake releases energy , the transmembrane domain effectively powers the periplasmic domain to expel drugs and other molecules . Eicher et al . note that a similar mechanism has not been seen before in other efflux pumps or transporter proteins . Understanding how AcrB works opens up new avenues that could be exploited to develop new drugs against multidrug resistant bacteria . Furthermore , Eicher et al . suggest that efflux pumps in humans closely related to AcrB might function in a similar way—including those required for regulation of cellular cholesterol , and for the correct development of embryos .