Modulation of ATPase Activity by Physical Disengagement of the ATP-binding Domains of an ABC Transporter, the Histidine Permease
1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês
10.1074/jbc.274.26.18310
ISSN1083-351X
AutoresPeiqi Liu, Cheng E. Liu, Giovanna Ferro‐Luzzi Ames,
Tópico(s)Hemoglobinopathies and Related Disorders
ResumoThe membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703–707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters. The membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703–707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters. The ABC transporter superfamily (ATP-binding cassette transporters, also called traffic ATPases) comprises prokaryotic and eukaryotic ATP-energized transporters that share a conserved nucleotide-binding domain and have similar architectural organization (1Ames G.F.-L. Cell. 1986; 47: 323-324Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 2Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3344) Google Scholar). It constitutes the largest paralogous protein family in the fully sequenced genome of Escherichia coli andTreponema pallidum (3Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. ColladoVides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1460Crossref PubMed Scopus (5967) Google Scholar, 4Fraser C.M. Norris S.J. Weinstock G.M. White O. Sutton G.G. Dodson R. Gwinn M. Hickey E.K. Clayton R. Ketchum K.A. Sodergren E. Hardham J.M. McLeod M.P. Salzberg S. Peterson J. Khalak H. Richardson D. Howell J.K. Chidambaram M. Utterback T. McDonald L. Artiach P. Bowman C. Cotton M.D. Fujii C. Garland S. Hatch B. Horst K. Roberts K. Sandusky M. Weidman J. Smith H.O. Venter J.C. Science. 1998; 281: 375-388Crossref PubMed Scopus (834) Google Scholar). The superfamily includes prokaryotic periplasmic binding protein-dependent transporters (5Boos W. Lucht J.M. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1175-1209Google Scholar), such as the well characterized Salmonella typhimurium histidine permease, and many medically important transporters in humans, such as the cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domains; MOPS, 4-morpholinepropanesulfonic acid; HisQMP2, membrane-bound complex containing HisQ, HisM, and HisP; HisP*, constitutive mutant HisP; HisQM, the integral membrane proteins HisQ and HisM; HisQMP6His, purified HisQMP2 with a carboxyl-terminal extension to HisP of 8 amino acids residues: Leu-Glu-His-His-His-His-His-His; PLS, reconstituted proteoliposomes; ATPγS, adenosine 5′-O-(thiotriphosphate); AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; AMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate).1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domains; MOPS, 4-morpholinepropanesulfonic acid; HisQMP2, membrane-bound complex containing HisQ, HisM, and HisP; HisP*, constitutive mutant HisP; HisQM, the integral membrane proteins HisQ and HisM; HisQMP6His, purified HisQMP2 with a carboxyl-terminal extension to HisP of 8 amino acids residues: Leu-Glu-His-His-His-His-His-His; PLS, reconstituted proteoliposomes; ATPγS, adenosine 5′-O-(thiotriphosphate); AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; AMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate).(6Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5851) Google Scholar), the multidrug resistance protein (MDR or P-glycoprotein) (7Gottesman M.M. Pastan I. J. Biol. Chem. 1988; 263: 12163-12166Abstract Full Text PDF PubMed Google Scholar), the transporters associated with antigen processing (TAP1/TAP2) (8Trowsdale J. Hanson I. Mockridge I. Beck S. Townsend A. Kelly A. Nature. 1990; 348: 741-744Crossref PubMed Scopus (603) Google Scholar), and the photoreceptor cell-specific transporter ABCR (9Allikmets R. Singh N. Sun H. Shroyer N.F. Hutchinson A. Chidambaram A. Gerrard B. Baird L. Stauffer D. Peiffer A. Rattner A. Smallwood P. Li Y. Anderson K.L. Lewis R.A. Nathans J. Leppert M. Dean M. Lupski J.R. Nat. Genet. 1997; 15: 236-246Crossref PubMed Scopus (1085) Google Scholar). Prokaryotic and eukaryotic ABC transporters have the same overall topological organization, with two hydrophobic domains and two nucleotide-binding domains (NBDs). In prokaryotes these domains are usually composed of separate protein subunits, whereas in eukaryotes they are usually fused into a single polypeptide.The periplasmic histidine permease is an excellent model system to study the mechanism of action of the ABC transporter superfamily in general. It is composed of a soluble receptor, the periplasmic histidine-binding protein, HisJ, and a four-subunit membrane-bound complex (HisQMP2) composed of HisQ and HisM (the two hydrophobic subunits) and of two identical HisP subunits that carry the highly conserved nucleotide-binding domain (10Ames G.F.-L. Curr. Top. Membr. Transp. 1985; 23: 103-119Crossref Scopus (18) Google Scholar, 11Doige C.A. Ames G.F.-L. Annu. Rev. Microbiol. 1993; 47: 291-319Crossref PubMed Scopus (265) Google Scholar). The membrane-bound complex of the histidine permease has been purified and reconstituted into proteoliposomes (PLS), and shown to hydrolyze ATP and translocate histidine in a manner that is strictly dependent on the presence of the binding protein (12Liu C.E. Ames G.F.-L. J. Biol. Chem. 1997; 272: 859-866Crossref PubMed Scopus (49) Google Scholar). The membrane-bound complex must interact with the liganded soluble receptor in order to achieve translocation (13Ames G.F.-L. Noel K.D. Taber H. Spudich E.N. Nikaido K. Afong J. Ardeshir F. J. Bacteriol. 1977; 129: 1289-1297Crossref PubMed Google Scholar, 14Ames G.F.-L. Lever J. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 1096-1103Crossref PubMed Scopus (71) Google Scholar). In the absence of the binding protein, the complex hydrolyzes ATP at a low rate (intrinsic activity), which is stimulated by the liganded or unliganded binding protein; the level of stimulation is highest when HisJ is liganded (15Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Crossref PubMed Scopus (111) Google Scholar).Numerous lines of evidence suggest that ATP binding and hydrolysis is performed by the NBDs, HisP in the case of the histidine permease, with the hydrophobic subunits controlling the rate of hydrolysis. HisP is photolabeled by azido-ATP analogs (16Hobson A.C. Weatherwax R. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7333-7337Crossref PubMed Scopus (70) Google Scholar); mutations in the ATP-binding pocket lose the ability to bind azido-ATP (17Shyamala V. Baichwal V. Beall E. Ames G.F.-L. J. Biol. Chem. 1991; 266: 18714-18719Abstract Full Text PDF PubMed Google Scholar); the membrane complex does not hydrolyze ATP when depleted of HisP, and hydrolysis is resumed after purified HisP is reconstituted back into the complex (18Liu P.-Q. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3495-3500Crossref PubMed Scopus (48) Google Scholar); and purified soluble HisP hydrolyzes ATP in the absence of HisQ and HisM (19Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The recent resolution of the three-dimensional structure of HisP at 1.5 Å clearly shows the presence of an ATP-binding pocket containing an ATP molecule (20Hung L.-W. Wang I.X. Nikaido K. Liu P.-Q. Ames G.F.-L. Kim S.-H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar).HisP has a hydrophilic sequence, yet it is tightly associated with the membrane; however, it behaves neither like a classical integral membrane protein nor like a peripheral membrane protein (21Kerppola R.E. Shyamala V.K. Klebba P. Ames G.F.-L. J. Biol. Chem. 1991; 266: 9857-9865Abstract Full Text PDF PubMed Google Scholar). HisP is accessible to proteases and impermeant biotinylating reagents from both the cytoplasmic and the periplasmic sides of the membrane, indicating that it spans the lipid bilayer (22Baichwal V. Liu D. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 620-624Crossref PubMed Scopus (68) Google Scholar). This membrane-spanning property has also been demonstrated for other ABC transporters, such as MalK (23Schneider E. Hunke S. Tebbe S. J. Bacteriol. 1995; 177: 5364-5367Crossref PubMed Google Scholar), the ATP-binding domain of CFTR (24Ko Y.H. Delannoy M. Pedersen P.L. Biochemistry. 1997; 36: 5053-5064Crossref PubMed Scopus (25) Google Scholar, 25Gruis D.B. Price E.M. Biochemistry. 1997; 36: 7739-7745Crossref PubMed Scopus (23) Google Scholar), and KpsT (26Bliss J.M. Silver R.P. J. Bacteriol. 1997; 179: 1400-1403Crossref PubMed Google Scholar). Because of the hydrophilic sequence of HisP, its membrane-spanning properties are likely to depend on a close interaction with the integral membrane subunits, HisQ and HisM (HisQM), with the strength of this interaction being important for regulating its ATPase activity. In this respect, there is evidence that HisQ and HisM are involved in the transmission of the transmembrane signal initiated by the soluble periplasmic receptor, HisJ, and in the stimulation of ATP hydrolysis by HisP (18Liu P.-Q. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3495-3500Crossref PubMed Scopus (48) Google Scholar,27Petronilli V. Ames G.F.-L. J. Biol. Chem. 1991; 266: 16293-16296Abstract Full Text PDF PubMed Google Scholar). Physical contact between HisJ and HisQ has been demonstrated by chemical cross-linking experiments (28Ames G.F.L. Liu C.E. Joshi A.K. Nikaido K. J. Biol. Chem. 1996; 271: 14264-14270Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Direct contact between HisP and HisJ is not expected and was not found.Permease mutations in the ATP-binding subunit (HisP*) were characterized that allow ligand translocation (29Ames G.F.-L. Spudich E.N. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1877-1881Crossref PubMed Scopus (51) Google Scholar, 30Speiser D.M. Ames G.F.-L. J. Bacteriol. 1991; 173: 1444-1451Crossref PubMed Google Scholar) and ATP hydrolysis in the absence of the binding protein (27Petronilli V. Ames G.F.-L. J. Biol. Chem. 1991; 266: 16293-16296Abstract Full Text PDF PubMed Google Scholar), indicating an altered signaling mechanism. To understand further these mechanisms and their regulation during a functional cycle, the biochemical properties of these HisP* mutants were analyzed in detail, and several novel characteristics were defined. HisQMP*2 complexes have a looser structure, with the mutant HisP* being "disengaged" from HisQM; they have lower affinity for ADP than the wild type; and their ATPase activity displays no cooperativity for ATP. Based on these observations, we propose a mechanistic model for ATP hydrolysis and ligand translocation which may be applicable to other ABC transporters.DISCUSSIONWe show that the interaction between the ATP-binding subunit (HisP) and the integral transmembrane subunits (HisQ and HisM) is weaker in mutant HisQMP*2 complexes than in the wild type, indicating that the integrity of the complex is compromised by these mutations. We refer to this phenomenon as "disengagement of HisP from HisQM." Concomitantly, these mutant complexes display a higher rate of constitutive ATP hydrolysis. The behavior of these mutant HisP*s resembles that of peripheral membrane proteins, so that they could be viewed as interaction mutants. Such a notion is consistent with the recent resolution of the crystal structure of HisP, which was shown to have an "L" shape, with two separate arms: arm I is mostly dedicated to forming the ATP-binding pocket, whereas arm II is likely to form the domain that interacts physically with HisQM (20Hung L.-W. Wang I.X. Nikaido K. Liu P.-Q. Ames G.F.-L. Kim S.-H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar). The formation of the correct quaternary structure of the complex would be expected to be dependent upon the nature of the interaction between arm II of HisP and HisQM. Such a quaternary structure is likely to be essential not only for the correct enzymatic activity of the complex but also for creating the signaling pathway that regulates its ATPase activity. The fact that all of the mutations considered in this paper, which simultaneously result in a looser complex structure and in signal-independent ATPase activity, are clustered and located in arm II is consistent with the idea that arm II interacts with HisQM and that the mutated residues are involved either directly or indirectly in an interaction with HisQ and/or HisM.If the hypothesis of an interaction between arm II and HisQM is correct, it should be possible to identify mutants in arm II that have completely eliminated the interaction between HisP and HisQM, resulting in a complex that is altogether unable to insert into the membrane. Mutants of this type are likely to exist but would have gone unrecognized through our selection procedures because they would be inactive in transport activity. It is worthwhile stressing the fact that all of the mutations analyzed here had been isolated with the requirement that transport activity be maintained (in the absence of the periplasmic binding protein). Thus, they all have modified the function of the complex but have not inactivated it. It also should be mentioned that mutations with properties equivalent to those of HisP* mutants but located in the hydrophobic subunits should also exist. Likely candidates indeed have been identified both in HisQ 4G. F.-L. Ames, unpublished results. and in the hydrophobic components of the maltose permease (38Covitz K.M. 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).The observation that HisP* is disengaged has important implications for the mechanism of ATPase activation. ATP hydrolysis by HisP during the transport cycle is regulated by HisQ and HisM through a balanced counterforce of stimulation and suppression (15Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Crossref PubMed Scopus (111) Google Scholar, 18Liu P.-Q. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3495-3500Crossref PubMed Scopus (48) Google Scholar, 19Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). HisQM, through its physical association with HisP, would exercise such a dual role and regulate the ATPase activity, thus preventing useless ATP consumption and promoting a swift response when a ligand is presented. During a cycle of ATP hydrolysis, HisQ and HisM may alternate between an activating and a suppressing mode, with HisQM assuming the activating mode when liganded HisJ is present and the suppressing mode in the absence of HisJ. The fact that a tight complex structure is associated with low ATPase activity suggests that engagement of HisP leads to suppression of the ATPase. Such a suppression (when the complex is in the resting stage, see below) could be exercised in a variety of ways. HisP might come in direct contact with phospholipids that inhibit the ATPase activity (19Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), or its nucleotide-binding pocket may be disrupted or not be fully accessible, or HisQM might increase the affinity for ADP, thus slowing down the rate of ADP release (HisQMP2 indeed has higher affinity for ADP than soluble HisP, with IC50 values of 0.1 versus 0.7 mm ADP, respectively).The proposed cycle of membrane disengagement/reengagement is reminiscent of SecA, the E. coli translocation ATPase, which undergoes ATP-dependent membrane insertion and deinsertion (39Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar). However, ATP binding and hydrolysis have been modeled to achieve different goals in the process of membrane cycling. Upon ATP hydrolysis, SecA releases the preprotein peptide and deinserts from the membrane, whereas in the case of HisP, ATP binding causes the disengagement of HisP and reengagement occurs after hydrolysis. The fundamental difference between HisP and SecA may be related to the difference in transport direction: SecA translocates peptides from inside to outside (39Economou A. Wickner W. Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 40Shinkai A. Mei L.H. Tokuda H. Mizushima S. J. Biol. Chem. 1991; 266: 5827-5833Abstract Full Text PDF PubMed Google Scholar, 41Kim Y.J. Rajapandi T. Oliver D. Cell. 1994; 78: 845-853Abstract Full Text PDF PubMed Scopus (146) Google Scholar), whereas the histidine permease translocates substrate from outside to inside.The fact that the ATPase activity of wild type HisQMP2shows positive cooperativity for ATP, whereas that of the HisQMP* mutants does not, provides some insight into the mechanism of ATP hydrolysis. Since such cooperativity is observed both for the HisJ-stimulated and intrinsic activities (15Liu C.E. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Crossref PubMed Scopus (111) Google Scholar), it must be independent of HisJ signaling. Because mutant HisQMP*2 is already disengaged in the absence of ATP and shows no cooperativity for ATP, the positive cooperativity observed for the wild type may be explained by an initial binding of ATP to a low affinity site that induces HisP disengagement, with the disengaged complex subsequently binding the second ATP with higher affinity. It is also relevant that HisP dimer in the soluble form, which can be viewed as fully disengaged, also shows no cooperativity (19Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The two identical HisP molecules within a complex may acquire different ATP-binding and -hydrolyzing properties because of an asymmetric interaction with the two different subunits, HisQ and HisM. It should be noted, however, that the two cooperative binding sites could also be one in HisQ and the other in one of the HisP subunits, since HisQ is known to bind ATP (16Hobson A.C. Weatherwax R. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7333-7337Crossref PubMed Scopus (70) Google Scholar,42Ames G.F. Mimura C.S. Holbrook S.R. Shyamala V. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 1-47PubMed Google Scholar). 5V. Shyamala and G. F.-L. Ames, unpublished data. The lack of cooperativity in HisQMP*2 is in contrast with the results presented in the equivalent type of binding protein-independent mutants in the maltose system, which retain cooperativity (43Davidson A.L. Laghaeian S.S. Mannering D.E. J. Biol. Chem. 1996; 271: 4858-4863Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The reason for this discrepancy is not clear. It is possible, although we consider it unlikely, that the two permeases differ in the mechanism of transport; alternatively, the fact that the mutations causing constitutivity occur in a hydrophobic subunit in the maltose system and in the ATP-binding subunit in the histidine system might result in different behavior.Although HisP disengagement appears to be a priming mechanism for ATPase activation, it is not sufficient to activate ATP hydrolysis. The presence of ATP alone disengages wild type HisP but does not result in ATP hydrolysis in the absence of HisJ. Hydrolysis is not necessary for disengagement because several mutant complexes that bind ATP normally, but cannot hydrolyze it, display an ATP-induced disengagement (as measured by urea extraction) identical to that of wild type HisP (data not shown). Although HisJ stimulates ATP hydrolysis, it was not found to induce HisP disengagement by itself. Soluble HisP (i.e.fully disengaged) does not hydrolyze ATP at a rate as high as its potential, i.e. as high as that of fully stimulated HisQMP2 (19Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Therefore, there must be other events or determining factors regulating the rate of ATP hydrolysis by the wild type complex. The fact that soluble HisP* mutants hydrolyze ATP at a higher rate than soluble wild type HisP (Table II) suggests that this other factor(s) reflects an intrinsic property of HisP. A possible factor is suggested by the finding that the mutants have a poorer affinity for ADP than the wild type, both in the soluble and complex form (Table II); thus, their higher level of ATP hydrolysis may result from an accelerated rate of ADP release.In light of our results, we propose a revised model for binding protein-dependent ABC transporters (Fig.9). In the resting stage (stage I) HisP is engaged and inactive. Upon binding ATP, the complex assumes a different quaternary structure, in which the ATP-binding subunit becomes loosely attached (disengaged; stage II). ATP hydrolysis follows upon signaling by the soluble receptor, which alters the conformation of HisQM (stage III), followed by the opening of a passageway within the complex thus allowing substrate translocation (stage IV). In stage II, despite disengagement of HisP, ATP is hydrolyzed at a low rate (intrinsic ATPase activity), because HisQM is still in the suppression mode and HisP has a high affinity for ADP, which is therefore released slowly. In stage III, the liganded binding protein converts HisQM to an activating mode, which enables rapid ADP release. HisQMP*2mutants already have a disengaged structure in the resting stage, in which HisP* mimics stage III of the wild type even in the absence of the binding protein and with HisQM in the stage I/II conformation. These mutants are always in a primed state for hydrolysis, because HisP* is constantly in an activated form and has a lower affinity for ADP.Although this model provides a mechanism specific for the binding protein-dependent ABC transporters, these results could be extrapolated to other members of the superfamily. This may be particularly true because the structure of the HisQMP2* mutants is reminiscent of that of eukaryotic ABC transporters in that neither relies on a soluble receptor for ATP hydrolysis and transport. In this respect it is interesting to compare the properties of HisQMP*2 mutants with those of the most common mutation, ΔPhe-508, in CFTR in which a phenylalanine residue (Phe-508 in NBD1) has been deleted. Upon alignment of the sequence of CFTR with that of HisP, Phe-508 is found to be located in a position in the crystal structure very similar to that of the HisP* mutants described here (20Hung L.-W. Wang I.X. Nikaido K. Liu P.-Q. Ames G.F.-L. Kim S.-H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar). Thus, a reasonable hypothesis is that ΔPhe-508 is defective in the interaction between NBD1 and its respective hydrophobic domain(s), and as a consequence its overall folded structure is incorrect. This proposal is compatible with the finding that among the characteristics of ΔPhe-508 are its inability to reach its final site of action and the fact that the defect can be obviated by protein-stabilizing conditions, such as the presence of glycerol or growing the cells producing ΔPhe-508 at low temperature (44Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1405) Google Scholar, 45Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 358: 761-764Crossref PubMed Scopus (1054) Google Scholar, 46Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 47Qu B.H. Strickland E. Thomas P.J. J. Bioenerg. Biomemb. 1997; 29: 483-490Crossref PubMed Scopus (52) Google Scholar). Presumably, the formation of a defective folded structure during CFTR synthesis affects its insertion into the membrane and results in mislocation of the protein. It should be stressed that the defect of ΔPhe-508 is not in its activity and that this situation is similar to that of HisP* mutants which, although defective in the structure of the complex, are still functional in ATP hydrolysis and transport (27Petronilli V. Ames G.F.-L. J. Biol. Chem. 1991; 266: 16293-16296Abstract Full Text PDF PubMed Google Scholar, 30Speiser D.M. Ames G.F.-L. J. Bacteriol. 1991; 173: 1444-1451Crossref PubMed Google Scholar). The ABC transporter superfamily (ATP-binding cassette transporters, also called traffic ATPases) comprises prokaryotic and eukaryotic ATP-energized transporters that share a conserved nucleotide-binding domain and have similar architectural organization (1Ames G.F.-L. Cell. 1986; 47: 323-324Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 2Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3344) Google Scholar). It constitutes the largest paralogous protein family in the fully sequenced genome of Escherichia coli andTreponema pallidum (3Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. ColladoVides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1460Crossref PubMed Scopus (5967) Google Scholar, 4Fraser C.M. Norris S.J. Weinstock G.M. White O. Sutton G.G. Dodson R. Gwinn M. Hickey E.K. Clayton R. Ketchum K.A. Sodergren E. Hardham J.M. McLeod M.P. Salzberg S. Peterson J. Khalak H. Richardson D. Howell J.K. Chidambaram M. Utterback T. McDonald L. Artiach P. Bowman C. Cotton M.D. Fujii C. Garland S. Hatch B. Horst K. Roberts K. Sandusky M. Weidman J. Smith H.O. Venter J.C. Science. 1998; 281: 375-388Crossref PubMed Scopus (834) Google Scholar). The superfamily includes prokaryotic periplasmic binding protein-dependent transporters (5Boos W. Lucht J.M. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1175-1209Google Scholar), such as the well characterized Salmonella typhimurium histidine permease, and many medically important transporters in humans, such as the cystic fibrosis transmembrane conductance regulator (CFTR) 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide-binding domains; MOPS, 4-morpholinepropanesulfonic acid; HisQMP2, membrane-bound complex containing HisQ, HisM, and HisP; HisP*, constitutive mutant HisP; HisQM, the integral membrane proteins HisQ and HisM; HisQMP6His, purified HisQMP2 with a carboxyl-terminal extension to HisP of 8 amino acids residues: Leu-Glu-His-His-His-His
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