Editing ABC transporter (section)

== Mechanism of transport ==
ABC transporters are [[active transport]]ers, that is, they use energy in the form of adenosine triphosphate (ATP) to translocate substrates across cell membranes. These proteins harness the energy of ATP binding and/or hydrolysis to drive conformational changes in the ''transmembrane domain (TMD)'' and consequently transport molecules.<ref name=hollenstein>{{cite journal | vauthors = Hollenstein K, Dawson RJ, Locher KP | title = Structure and mechanism of ABC transporter proteins | journal = Current Opinion in Structural Biology | volume = 17 | issue = 4 | pages = 412–8 | date = Aug 2007 | pmid = 17723295 | doi = 10.1016/j.sbi.2007.07.003 }}</ref> ABC importers and exporters have a common mechanism for transporting substrates. They are similar in their structures. The model that describes the conformational changes associated with the binding of the substrate is the ''alternating-access model''. In this model, the substrate binding site alternates between ''outward-'' and ''inward-facing conformations''. The relative binding affinities of the two conformations for the substrate largely determines the net direction of transport. For importers, since translocation is directed from the periplasm to the cytoplasm, the outward-facing conformation has higher binding affinity for the substrate. In contrast, the substrate binding affinity in exporters is greater in the inward-facing conformation.<ref name=rees/> A model that describes the conformational changes in the ''nucleotide-binding domain (NBD)'' as a result of ATP binding and hydrolysis is the ''ATP-switch model''. This model presents two principal conformations of the NBDs: formation of a closed dimer upon binding two ATP molecules and dissociation to an open dimer facilitated by ATP hydrolysis and release of inorganic [[phosphate]] (P<sub>i</sub>) and [[adenosine diphosphate]] (ADP). Switching between the open and closed dimer conformations induces conformational changes in the TMD resulting in substrate translocation.<ref name=higgins>{{cite journal | vauthors = Higgins CF, Linton KJ | title = The ATP switch model for ABC transporters | journal = Nature Structural & Molecular Biology | volume = 11 | issue = 10 | pages = 918–26 | date = Oct 2004 | pmid = 15452563 | doi = 10.1038/nsmb836 | s2cid = 23058653 }}</ref>

The general mechanism for the transport cycle of ABC transporters has not been fully elucidated, but substantial structural and biochemical data has accumulated to support a model in which ATP binding and hydrolysis is coupled to conformational changes in the transporter. The resting state of all ABC transporters has the NBDs in an open dimer configuration, with low affinity for ATP. This open conformation possesses a chamber accessible to the interior of the transporter. The transport cycle is initiated by binding of substrate to the high-affinity site on the TMDs, which induces conformational changes in the NBDs and enhances the binding of ATP. Two molecules of ATP bind, cooperatively, to form the closed dimer configuration. The closed NBD dimer induces a conformational change in the TMDs such that the TMD opens, forming a chamber with an opening opposite to that of the initial state. The affinity of the substrate to the TMD is reduced, thereby releasing the substrate. Hydrolysis of ATP follows and then sequential release of P<sub>i</sub> and then ADP restores the transporter to its basal configuration. Although a common mechanism has been suggested, the order of substrate binding, nucleotide binding and hydrolysis, and conformational changes, as well as interactions between the domains is still debated.<ref name=davidson/><ref name=davidsonchen/><ref name=pohl/><ref name=rees/><ref name=msbaward/><ref name=msbareyes2006/><ref name=hollenstein/><ref name=higgins/><ref name=locher2004>{{cite journal | vauthors = Locher KP | title = Structure and mechanism of ABC transporters | journal = Current Opinion in Structural Biology | volume = 14 | issue = 4 | pages = 426–31 | date = Aug 2004 | pmid = 15313236 | doi = 10.1016/j.sbi.2004.06.005 }}</ref><ref name=oldham2008>{{cite journal | vauthors = Oldham ML, Davidson AL, Chen J | title = Structural insights into ABC transporter mechanism | journal = Current Opinion in Structural Biology | volume = 18 | issue = 6 | pages = 726–33 | date = Dec 2008 | pmid = 18948194 | pmc = 2643341 | doi = 10.1016/j.sbi.2008.09.007 }}</ref><ref name=msbachang2003>{{cite journal | vauthors = Chang G | title = Multidrug resistance ABC transporters | journal = FEBS Letters | volume = 555 | issue = 1 | pages = 102–5 | date = Nov 2003 | pmid = 14630327 | doi = 10.1016/S0014-5793(03)01085-8 | s2cid = 24228062 | doi-access = free | bibcode = 2003FEBSL.555..102C }}</ref>

Several groups studying ABC transporters have differing assumptions on the driving force of transporter function. It is generally assumed that ATP hydrolysis provides the principal energy input or "power stroke" for transport and that the NBDs operate alternately and are possibly involved in different steps in the transport cycle.<ref name=senior>{{cite journal | vauthors = Senior AE, al-Shawi MK, Urbatsch IL | title = The catalytic cycle of P-glycoprotein | journal = FEBS Letters | volume = 377 | issue = 3 | pages = 285–9 | date = Dec 1995 | pmid = 8549739 | doi = 10.1016/0014-5793(95)01345-8 | s2cid = 20395778 | doi-access =  | bibcode = 1995FEBSL.377..285S }}</ref> However, recent structural and biochemical data shows that ATP binding, rather than ATP hydrolysis, provides the "power stroke".<ref>{{cite journal |last1=Simpson |first1=Brent W. |last2=Pahil |first2=Karanbir S. |last3=Owens |first3=Tristan W. |last4=Lundstedt |first4=Emily A. |last5=Davis |first5=Rebecca M. |last6=Kahne |first6=Daniel |last7=Ruiz |first7=Natividad |title=Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport |journal=mBio |date=20 August 2019 |volume=10 |issue=4 |pages=e01931–19, /mbio/10/4/mBio.01931–19.atom |doi=10.1128/mBio.01931-19|pmid=31431556 |pmc=6703430 |doi-access=free }}</ref> It may also be that since ATP binding triggers NBD dimerization, the formation of the dimer may represent the "power stroke." In addition, some transporters have NBDs that do not have similar abilities in binding and hydrolyzing ATP and that the interface of the NBD dimer consists of two ATP binding pockets suggests a concurrent function of the two NBDs in the transport cycle.<ref name=higgins/>

Some evidence to show that ATP binding is indeed the power stroke of the transport cycle was reported.<ref name=higgins/> It has been shown that ATP binding induces changes in the substrate-binding properties of the TMDs. The affinity of ABC transporters for substrates has been difficult to measure directly, and indirect measurements, for instance through stimulation of ATPase activity, often reflects other rate-limiting steps. Recently, direct measurement of [[vinblastine]] binding to [[permease]]-glycoprotein ([[P-glycoprotein]]) in the presence of nonhydrolyzable ATP analogs, e.g. 5'-adenylyl-β-γ-imidodiphosphate (AMP-PNP), showed that ATP binding, in the absence of hydrolysis, is sufficient to reduce substrate-binding affinity.<ref name="pmid11747450">{{cite journal | vauthors = Martin C, Higgins CF, Callaghan R | title = The vinblastine binding site adopts high- and low-affinity conformations during a transport cycle of P-glycoprotein | journal = Biochemistry | volume = 40 | issue = 51 | pages = 15733–42 | date = Dec 2001 | pmid = 11747450 | doi = 10.1021/bi011211z }}</ref> Also, ATP binding induces substantial conformational changes in the TMDs. [[Spectroscopy|Spectroscopic]], [[protease]] accessibility and [[Cross-link|crosslinking]] studies have shown that ATP binding to the NBDs induces conformational changes in multidrug resistance-associated protein-1 (MRP1),<ref name="pmid12424247">{{cite journal | vauthors = Manciu L, Chang XB, Buyse F, Hou YX, Gustot A, Riordan JR, Ruysschaert JM | title = Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione | journal = The Journal of Biological Chemistry | volume = 278 | issue = 5 | pages = 3347–56 | date = Jan 2003 | pmid = 12424247 | doi = 10.1074/jbc.M207963200 | doi-access = free }}</ref> HisPMQ,<ref name="pmid11087367">{{cite journal | vauthors = Kreimer DI, Chai KP, Ferro-Luzzi Ames G | title = Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex | journal = Biochemistry | volume = 39 | issue = 46 | pages = 14183–95 | date = Nov 2000 | pmid = 11087367 | doi = 10.1021/bi001066 }}</ref> LmrA,<ref name="pmid10753896">{{cite journal | vauthors = Vigano C, Margolles A, van Veen HW, Konings WN, Ruysschaert JM | title = Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis | journal = The Journal of Biological Chemistry | volume = 275 | issue = 15 | pages = 10962–7 | date = Apr 2000 | pmid = 10753896 | doi = 10.1074/jbc.275.15.10962 | s2cid = 33274934 | url = https://dipot.ulb.ac.be/dspace/bitstream/2013/77623/1/J_Biol_Chem_2000_275_15_10962.pdf | doi-access = free }}</ref> and Pgp.<ref name="pmid10364203">{{cite journal | vauthors = Sonveaux N, Vigano C, Shapiro AB, Ling V, Ruysschaert JM | title = Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence quenching analysis | journal = The Journal of Biological Chemistry | volume = 274 | issue = 25 | pages = 17649–54 | date = Jun 1999 | pmid = 10364203 | doi = 10.1074/jbc.274.25.17649 | doi-access = free }}</ref> Two dimensional crystal structures of AMP-PNP-bound Pgp showed that the major conformational change during the transport cycle occurs upon ATP binding and that subsequent ATP hydrolysis introduces more limited changes.<ref name="pmid11598005">{{cite journal | vauthors = Rosenberg MF, Velarde G, Ford RC, Martin C, Berridge G, Kerr ID, Callaghan R, Schmidlin A, Wooding C, Linton KJ, Higgins CF | title = Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle | journal = The EMBO Journal | volume = 20 | issue = 20 | pages = 5615–25 | date = Oct 2001 | pmid = 11598005 | pmc = 125677 | doi = 10.1093/emboj/20.20.5615 }}</ref> Rotation and tilting of transmembrane α-helices may both contribute to these conformational changes. Other studies have focused on confirming that ATP binding induces NBD closed dimer formation. Biochemical studies of intact transport complexes suggest that the conformational changes in the NBDs are relatively small. In the absence of ATP, the NBDs may be relatively flexible, but they do not involve a major reorientation of the NBDs with respect to the other domains. ATP binding induces a rigid body rotation of the two ABC subdomains with respect to each other, which allows the proper alignment of the nucleotide in the active site and interaction with the designated motifs. There is strong biochemical evidence that binding of two ATP molecules can be cooperative, that is, ATP must bind to the two active site pockets before the NBDs can dimerize and form the closed, catalytically active conformation.<ref name=higgins/>