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ATP-driven MalK Dimer Closure and Reopening and Conformational Changes of the “EAA” Motifs Are Crucial for Function of the Maltose ATP-binding Cassette Transporter (MalFGK2)

2007; Elsevier BV; Volume: 282; Issue: 31 Linguagem: Inglês

10.1074/jbc.m701979200

1083-351X

Martin L. Daus, Mathias Grote, Peter Muöller, Meike Doebber, Andreas Herrmann, Heinz‐Jürgen Steinhoff, Elie Dassa, Erwin Schneider,

Peroxisome Proliferator-Activated Receptors

We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK2) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model. We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK2) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model. ATP-binding cassette (ABC) 3The abbreviations used are: ABC, ATP-binding cassette; BPI, binding protein-independent maltose transporter; F*, Cys-less MalF subunit; G*, Cys-less MalG subunit; DTT, dithiothreitol; EBS, 1,2-ethanediyl-bis(methanethiosulfonate); HBS, 1,6-hexandiyl-bis(methanethiosulfonate); MIANS, 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid; NBD, nucleotide binding domain; PBS, 3,6,9,12,15-pentaoxaheptadecan-1,17-diyl-bis(methanethiosulfonate); TMD, transmembrane domain; CuPhe, Cu(1,10-phenanthroline)2SO4. 3The abbreviations used are: ABC, ATP-binding cassette; BPI, binding protein-independent maltose transporter; F*, Cys-less MalF subunit; G*, Cys-less MalG subunit; DTT, dithiothreitol; EBS, 1,2-ethanediyl-bis(methanethiosulfonate); HBS, 1,6-hexandiyl-bis(methanethiosulfonate); MIANS, 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid; NBD, nucleotide binding domain; PBS, 3,6,9,12,15-pentaoxaheptadecan-1,17-diyl-bis(methanethiosulfonate); TMD, transmembrane domain; CuPhe, Cu(1,10-phenanthroline)2SO4. transporters are involved in the uptake or export of an enormous variety of substances ranging from small ions to large polypeptides at the expense of ATP. They are found in all organisms from bacteria to humans, and dysfunction is often associated with disease in humans, such as cystic fibrosis, adrenoleukodystrophy, or Stargardt's macular dystrophy (1Holland E.B. Cole S. Kuchler K. Higgins C.F. ABC Proteins: From Bacteria to Man. 2003; (, Academic Press, Inc., New York)Google Scholar). ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains (TMDs) that form the translocation pathway and two hydrophilic nucleotide binding (ABC) domains (NBDs) that hydrolyze ATP. In fact, in most prokaryote importers, TMDs and NBDs are expressed as separate protein subunits, whereas in most export systems of both prokaryotes and eukaryotes, they are usually fused into a single polypeptide chain (2Dassa E. Holland E.B. Cole S. Kuchler K. Higgins C.F. ABC Proteins: From Bacteria to Man. 2003: 3-35Google Scholar). The ABC domains are characterized by a set of Walker A and B motifs that are involved in nucleotide binding and by the unique "LSGGQ" signature sequence (3Schneider E. Hunke S. FEMS Microbiol. Rev. 1998; 22: 1-20Crossref PubMed Google Scholar). The crystal structures of several mostly prokaryotic NBDs have been reported that largely agree on the overall fold (reviewed in Refs. 4Schmitt L. Tampé R. Curr. Opin. Struct. Biol. 2002; 12: 754-760Crossref PubMed Scopus (275) Google Scholar, 5Jones P.M. George A.M. Cell Mol. Life Sci. 2004; 61 (.): 682-699Crossref PubMed Scopus (448) Google Scholar, 6Davidson A.L. Chen J. Annu. Rev. Biochem. 2004; 73: 241-268Crossref PubMed Scopus (487) Google Scholar). Accordingly, the cassette can be divided into a RecA-like subdomain encompassing both Walker motifs and a specific α-helical subdomain comprising the LSGGQ motif. Both subdomains are joined by the Q-loop containing a conserved glutamine residue that binds to the Mg2+ ion and attacking water. Structural and biochemical evidence have further revealed that in the physiologically relevant NBD dimer, the nucleotide binding site of one monomer is completed by the LSGGQ motif of the opposing monomer (7Hopfner K-N. Karcher A. Shin D.S. Craig L. Arthur L.M. Carney J.P. Tainer J.A. Cell. 2000; 101: 789-800Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 8Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 10Zaitseva J. Jenewein S. Jumpertz T. Holland B.I. Schmitt L. EMBO J. 2005; 24: 1901-1910Crossref PubMed Scopus (284) Google Scholar, 11Fetch E.E. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9685-9690Crossref PubMed Scopus (118) Google Scholar, 12Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Further progress in understanding the structural organization of ABC transporters was achieved with the high resolution structures of complete transporters (13Locher K. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (930) Google Scholar, 14Dawson R.J.P. Locher K.P. Nature. 2006; 443: 180-185Crossref PubMed Scopus (1072) Google Scholar, 15Pinkett H.W. Lee A.T. Lum P. Locher K.P. Rees D.C. Science. 2007; 315: 373-377Crossref PubMed Scopus (240) Google Scholar, 16Hollenstein K. Frei D.C. Locher K.P. Nature. 2007; 446: 213-216Crossref PubMed Scopus (400) Google Scholar). These structures have provided some insight into the interactions between the NBDs and the TMDs, although the means by which ATP-dependent conformational changes of the ABC domains are transmitted to the TMDs to affect substrate translocation are still poorly understood (5Jones P.M. George A.M. Cell Mol. Life Sci. 2004; 61 (.): 682-699Crossref PubMed Scopus (448) Google Scholar). The ABC transporter mediating the uptake of maltose and maltodextrins in Escherichia coli/Salmonella typhimurium is one of the best characterized systems and thus serves as a model for studying the molecular mechanism by which ABC importers exert their functions (reviewed in Refs. 6Davidson A.L. Chen J. Annu. Rev. Biochem. 2004; 73: 241-268Crossref PubMed Scopus (487) Google Scholar and 17Schneider E. Holland E.B. Cole S. Kuchler K. Higgins C.F. ABC Proteins: From Bacteria to Man. 2003: 157-185Google Scholar). The transporter is composed of an extracellular (periplasmic) receptor, the maltose-binding protein (MalE), and a membrane-bound complex (MalFGK2), comprising the pore-forming hydrophobic subunits, MalF and MalG, and two copies of the ABC subunit, MalK. Association of the MalK subunits with MalF and MalG requires the so-called "EAA" sequence motifs (consensus EAAX3GX9IXLP) that are conserved in the last cytoplasmic loop of TMDs of ABC import systems (18Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar, 19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). MalK and closely related ABC subunits belonging to the CUT1 and MOI subfamilies of ABC importers (2Dassa E. Holland E.B. Cole S. Kuchler K. Higgins C.F. ABC Proteins: From Bacteria to Man. 2003: 3-35Google Scholar, 20Schneider E. Res. Microbiol. 2001; 152: 303-310Crossref PubMed Scopus (111) Google Scholar) contain a unique C-terminal extension, which in the crystal structures of MalK (9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) and CysA (21Scheffel F. Demmer U. Warkentin E. Huölsmann A. Schneider E. Ermler U. FEBS Lett. 2005; 579: 2953-2958Crossref PubMed Scopus (32) Google Scholar) contribute substantially to monomer-monomer contacts. The MalK structures further revealed that binding of ATP causes the nucleotide binding domains to close (9Chen J. Lu G. Lin J. Davidson A.L. Quiocho F.A. Mol. Cell. 2003; 12: 651-661Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) and that ATP hydrolysis is required for resetting the system to the resting state (22Lu G. Westbrooks J.M. Davidson A.L. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17969-17974Crossref PubMed Scopus (112) Google Scholar). Closing of the MalK dimer in solution is consistent with cross-linking data obtained with the intact transporter in crude membrane vesicles (19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Genetic and biochemical evidence has given rise to the notion that ATP binding to the MalK dimer might be communicated via its Q-loops to the EAA motifs of MalFG (18Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar, 19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In support of this view, the crystal structure of the vitamin B12 transporter of E. coli (BtuCD) not only revealed close contact between the Q-loop of BtuD and the EAA (or L) loops of BtuC but also contacts of the Q-loop with atoms in the ATP binding site (13Locher K. Lee A.T. Rees D.C. Science. 2002; 296: 1091-1098Crossref PubMed Scopus (930) Google Scholar). Furthermore, a molecular dynamics simulation suggests that conformational switching of the Q-loop may mediate communication between the TMDs and the catalytic sites (5Jones P.M. George A.M. Cell Mol. Life Sci. 2004; 61 (.): 682-699Crossref PubMed Scopus (448) Google Scholar). These and other data have led to a model according to which transport of maltose is initiated by interaction of liganded MalE (23Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (236) Google Scholar) with the nucleotide-free MalFGK2 complex at the extracellular (periplasmic) side, causing ATP binding to the MalK subunits and subsequent closing of the MalK dimer. ATP-driven dimer formation is thought to represents one possibility for the power stroke of ABC transporters (8Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar). The monomer-dimer transition would be coupled to conformational changes in the transmembrane domains for which evidence has recently been presented (24Daus M.L. Landmesser H. Schlosser A. Muöller P. Herrmann A. Schneider E. J. Biol. Chem. 2006; 281: 3856-3865Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Hydrolysis of one ATP molecule might result in simultaneous opening of the binding protein (25Chen J. Sharma S. Quiocho F.A. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1525-1530Crossref PubMed Scopus (177) Google Scholar) and of a gate at the periplasmic side, thereby giving the substrate molecule access to a translocation pathway through the membrane. Hydrolysis of the second ATP would bring the system back to the resting state. The concomitant separation of the NBDs is discussed as a second possibility for the power stroke of the system (8Smith P.C. Karpowich N. Millen L. Moody J.E. Rosen J. Thomas P.J. Hunt J.F. Mol. Cell. 2002; 10: 139-149Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar). In this paper, we have investigated ATP-dependent conformational changes of the purified MalFGK2 complex. By employing site-directed cross-linking, we determined distance changes between Ala-85 in both Q-loops of the MalK subunits upon the addition of ATP (Fig. 1A). The observed motional changes were used as "molecular pointers" to characterize the conformational state of transport-defective and binding protein-independent variants. The functional consequences of covalently linking the Q-loops to each other or the EAA motifs of MalFG to the helical subdomain of MalK (Fig. 1, A and B) were analyzed by monitoring MalE/maltose-stimulated ATPase activity of the complexes. Our results suggest that closing and reopening of the MalK dimer and conformational changes of the EAA motifs are crucial features within the catalytic cycle of the intact transporter. Bacterial Strains and Plasmids—E. coli strain JM109 (Stratagene) was used as a general host for the plasmids listed in Table 1. The plasmid-borne malK alleles originate from S. typhimurium, whereas the malFmalG alleles on pTZ18R are from E. coli. The mal genes from both organisms are functionally fully exchangeable (19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar).TABLE 1Plasmids used in this studyPlasmidRelevant genotype/DescriptionSource or referencemalFGKpBB1pT5 ampr/(MalFG His6-MalK) (wild type)28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google ScholarmalKpJS08pT5 camr /(His6-MalK)24Daus M.L. Landmesser H. Schlosser A. Muöller P. Herrmann A. Schneider E. J. Biol. Chem. 2006; 281: 3856-3865Abstract Full Text Full Text PDF PubMed Scopus (41) Google ScholarpMM37pT5 camr /(His6-MalK-C40S)24Daus M.L. Landmesser H. Schlosser A. Muöller P. Herrmann A. Schneider E. J. Biol. Chem. 2006; 281: 3856-3865Abstract Full Text Full Text PDF PubMed Scopus (41) Google ScholarpMM40pT5 camr /(His6-MalK-C40S,V117C)This studypMM41pT5 camr /(His6-MalK-C40S,A85C,Q159E)This studypMG13pT5 camr /(His6-MalK-C40S,A85C)This studypMG27pT5 camr /(His6-MalK-C40S,A85C,Q140K)This studypSH25pTrc ampr /(His6-MalK-C40S)26Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (11) Google ScholarmalFGpTAZFGQ*ptac ampr malF(Cys-)/malG(Cys-)19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarpTAZFGQ(S3C/-)ptac ampr malF (S403C)/malG(Cys-)19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarpTAZFGQ(-/A3C)ptac ampr malF (Cys-)/malG (A192C)19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarpMM20ptacampr malF(Cys-/G338R/N505I)malG(Cys-)This study Open table in a new tab Plasmid Constructions—Plasmid pMM37 was constructed by replacing an EcoRV fragment encompassing codons 25–292 of malK from pJS08 (24Daus M.L. Landmesser H. Schlosser A. Muöller P. Herrmann A. Schneider E. J. Biol. Chem. 2006; 281: 3856-3865Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) by the corresponding fragment from plasmid pSH25 (26Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (11) Google Scholar) harboring the malK796 (C40S) allele (Table 1). Cysteine residues were introduced by the Stratagene QuikChange kit using plasmid pTAZFGQ* (malF(Cys-)malG-(Cys-)) (19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) and pMM37 (malK796 (C40S)) as templates. Plasmid pMM20 was constructed by introducing the mutations G338R and N505I, conferring the binding protein-independent phenotype (27Covitz K-M. Y. Panagiotidis C.H Hor L.-I. Reyes M. Treptow N.A. Shuman H.A. EMBO J. 1994; 13: 1752-1759Crossref PubMed Scopus (65) Google Scholar) into the malF allele on plasmid pTAZFGQ* (19Hunke S. Mourez M. Jéhanno M. Dassa E. Schneider E. J. Biol. Chem. 2000; 275: 15526-15534Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) using Stratagene's QuikChange kit. Purification of MalFGK2 Complexes—Polyhistidine-tagged complex variants were overproduced in strain JM109, harboring the plasmids described in Table 2. Purification was essentially carried out as described in Ref. 28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar.TABLE 2Combination of plasmids used to overproduce the indicated complex variantsComplex variantmalKmalFGMalKFG (wild type)pBB1pBB1MalK(C40S)F*G*pMM37pTAZFGQ*MalK(C40S, A85C)F*G*pMG13pTAZFGQ*MalK(C40S, A85C)F*(S403C)G*pMG13pTAZFGQ(S3C/—)MalK(C40S, A85C)F*G*(A192C)pMG13pTAZFGQ(—/A3C)MalK(C40S, A85C)F500*G*pMG13pMM20MalK(C40S, A85C, E159Q)F*G*pMM41pTAZFGQ*MalK(C40S, A85C, Q140K)F*G*pMG27pTAZFGQ*MalK(C40S, V117C)F*(S403C)G*pMM40pTAZFGQ(S3C/—)MalK(C40S, V117C)F*G*(A192C)pMM40pTAZFGQ(—/A3C) Open table in a new tab Purification of MalE—Maltose-binding protein (tagless) was purified as described in Ref. 28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar. 2-(4′-Maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) Modification of Transport Complexes—MIANS was purchased from Molecular Probes (via Invitrogen). For MIANS modification studies, transport complexes were labeled in buffer 1 (50 mm Tris-HCl, pH 8, 20% glycerol, 0.01% β-d-dodecylmaltoside) by incubation at 4 °C with a 4-fold molar excess of MIANS for 15 min. The reaction was terminated by adding 1 mm dithiothreitol (DTT), and excess MIANS was removed from the protein sample by a PD10 desalting column (Amersham Biosciences) or by Ni2+-nitrilotriacetic acid affinity chromatography. Preparation of Vanadate-trapped Complexes—Wild type and mutant transport complexes were treated with vanadate as described in Ref. 25Chen J. Sharma S. Quiocho F.A. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1525-1530Crossref PubMed Scopus (177) Google Scholar. Briefly, vanadate was added to a final concentration of 0.5 mm to a mixture containing 2,5 μm MalFGK2 complex, 5 μm MalE, 4 mm ATP, 10 mm MgCl2, and 0.01 mm maltose in buffer 1, and the reaction was incubated for 20 min at 37 °C. For MIANS labeling, the samples were desalted by passage through a PD10 column. Stable association of MalE with the trapped complexes was confirmed using Ni2+-nitrilotriacetic acid affinity chromatography. Preparation of Proteoliposomes—MalE/maltose-loaded proteoliposomes were prepared as in Ref. 28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar. When cross-linked complexes were incorporated into liposomes, DTT was omitted from all buffers. Cross-linking with Cu(1,10-phenanthroline)2SO4 (CuPhe)—Complex protein (2.5 μm) was incubated in buffer 1 with 3 mm CuSO4 and 9 mm 1,10-phenanthroline (from Sigma) for 20 min at room temperature. Reactions were terminated by adding 5 mm N-ethylmaleimide. For ATPase measurements of cross-linked complexes, samples were passed through a PD-10 desalting column to remove CuPhe. Samples treated with DTT were also passed through PD 10 columns. For experiments with proteoliposomes protein samples were cross-linked prior to reconstitution. Cross-linking with Homobifunctional Sulfonate Cross-linkers—Sulfonate cross-linkers were purchased from Toronto Chemicals (Toronto, Canada). The following cross-linkers were used: 1,2-ethanediyl-bis(methanethiosulfonate) (EBS), 1,6-hexandiyl-bis(methanethiosulfonate) (HBS), and 3,6,9,12,15-pentaoxaheptadecan-1,17-diyl-bis(methanethiosulfonate) (PBS). According to Ref. 29Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, the approximate spacer lengths are 5.2 Å (EBS), 10.4 Å (HBS), and 24.7 Å (PBS). Reactions were started by adding the respective cross-linker (final concentration 1 mm) from freshly prepared stock solutions (100 mm in dimethyl sulfoxide) to the indicated protein samples (2.5 μm) in buffer 1. After 20 min at room temperature, reactions were terminated by adding 5 mm N-ethylmaleimide. ATPase Assay—Hydrolysis of ATP was assayed in microtiter plates essentially as described in Ref. 28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar. Protein Determination—Protein concentrations were determined by the method of Dulley and Grieve (30Dulley J.R. Grieve P.A. Anal. Biochem. 1975; 64: 136-141Crossref PubMed Scopus (852) Google Scholar). Electrophoresis—SDS-PAGE was performed with 10% polyacrylamide gels as described in Ref. 28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar. Fluorescent bands on gels were visualized in distilled water over an ultraviolet light source and recorded using a Bio-Rad Gel Doc EQ image system (Munich, Germany). Quantification of protein bands by densitometric scanning was carried using the GelScan Pro 5.0 software (BioSciTec GmbH). The MalK(C40S)F*G* Complex Variant—In the initial stage of this work, we set out to perform the intended cross-linking experiments and functional analyses with the purified MalK(A85C) variant of an otherwise Cys-less transport complex. However, purification by an established protocol (28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar) turned out to be highly insufficient due to unforeseen poor overproduction. Then, during the course of labeling complex variants with the fluorophore MIANS, we observed that in wild type MalK only Cys-40 reacted with the fluorophore, whereas the remaining cysteine residues (Cys-350 and Cys-360) were virtually inaccessible to the label (see Fig. 4 in Ref. 24Daus M.L. Landmesser H. Schlosser A. Muöller P. Herrmann A. Schneider E. J. Biol. Chem. 2006; 281: 3856-3865Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The complex variant MalK(C40S)F*G* (asterisks denote Cys-less variants) could be overproduced and purified in amounts comparable with wild type and exhibited similar MalE/maltose-stimulated ATPase activity both in proteoliposomes and in detergent solution (see Table 3). Moreover, ATP hydrolysis was markedly inhibited (>80%) by vanadate (not shown). Vanadate as a phosphate analogue can lock the complex in a so-called "transition state" by trapping ADP in one binding site and thereby preventing a new cycle of hydrolysis (25Chen J. Sharma S. Quiocho F.A. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1525-1530Crossref PubMed Scopus (177) Google Scholar, 31Sharma S. Davidson A.L. J. Bacteriol. 2000; 182: 6570-6576Crossref PubMed Scopus (83) Google Scholar). It is well established that these properties can be taken as evidence for tight coupling of ATP hydrolysis to maltose transport (23Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (236) Google Scholar, 24Daus M.L. Landmesser H. Schlosser A. Muöller P. Herrmann A. Schneider E. J. Biol. Chem. 2006; 281: 3856-3865Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 25Chen J. Sharma S. Quiocho F.A. Davidson A.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1525-1530Crossref PubMed Scopus (177) Google Scholar, 28Landmesser H. Stein A. Bluöschke B. Brinkmann M. Hunke S. Schneider E. Biochim. Biophys. Acta. 2002; 1565: 64-72Crossref PubMed Scopus (35) Google Scholar, 32Mannering D.E. Sharma S. Davidson A.L. J. Biol. Chem. 2001; 276: 12362-12368Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Together, we reasoned that it would be promising to construct the MalK variants studied in this work on the MalK(C40S) background.TABLE 3ATPase activities of maltose transport complex variantsComplex variantATPase activityaSpecific ATPase activities of purified transport complex variants in detergent solution (0.08 mg/ml) measured in the absence and presence of purified MalE (0.2 mg/ml) and maltose (10 μm).No addition+MalE/maltose+MalE/maltose + CuPhe+MalE/maltose + CuPhe + DTTμmol Pi/min/mgMalKFG (wild type)In liposomes0.111.3NDNDIn detergent solution0.060.35NDNDMalK(C40S)F*G*In liposomes0.131.11.0bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.1.0bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.In detergent solution0.020.310.320.31MalK(C40S,A85C)F*G*In liposomes0.180.740.22bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.0.68bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.In detergent solution0.060.170.080.13MalK(C40S,A85C,E159Q)F*G*In liposomes0.020.02NDNDIn detergent solution0.030.02NDNDMalK(C40S,A85C,Q140K)F*G*In detergent solution0.020.03NDNDMalK(C40S,A85C)F500*G*In liposomes0.60.76NDNDIn detergent solution0.440.52NDNDMalK(C40S,V117C)F*(S403C)G*In liposomes0.120.980.43bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.0.9bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.In detergent solution0.040.180.030.14MalK(C40S,V117C)F*G*(A192C)In liposomes01.10.21bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.0.92bComplexes were first cross-linked in detergent solution and subsequently incorporated into liposomes.In detergent solution0.210.650.230.59a Specific ATPase activities of purified transport complex variants in detergent solution (0.08 mg/ml) measured in the absence and presence of purified MalE (0.2 mg/ml) and maltose (10 μm).b Complexes were first cross-linked in detergent solution and subsequently incorporated into liposomes. Open table in a new tab ATP Binding Reduces the Distance between the Q-loops in the MalK Dimer—The purified complex variant MalK(C40S,A85C)F*G* was cross-linked in detergent solution in the absence of ATP, in an ATP-bound state, and after vanadate-trapping. We used homobifunctional sulfonate cross-linkers, covering distances of ∼5.2 Å (EBS), 10.4 Å (HBS), and 24.7 Å (PBS). As shown in Fig. 2A in the absence of ligand, a MalK dimer was formed only with PBS. In the presence of ATP, a cross-link product was observed with all three linkers, suggesting that the Ala-85 residues of both monomers have approached to a distance of apparently 5.2 Å. No cross-link products were found in the absence of chemical linkers (not shown). The observation that the cross-link reactions were significant but not complete might be due to limited temporal reactivity of the water-insoluble homobifunctional chemical linkers. Nonetheless, th