Title: The Crystal Structure of the MJ0796 ATP-binding Cassette
Abstract: The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-Å resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703–707) shows a 5-Å withdrawal of a phylogenetically invariant glutamine residue from contact with the γ-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F1-type ATP-binding core of the enzyme to an ABC transporter-specific α-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al.(2001)Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the α-helical subdomain as consistent consequences of γ-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel β-type hydrogen bonding interactions between the outermost β-strands in the two core β-sheets, leading to their fusion into a single extended β-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of β-sheet-containing proteins. The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-Å resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703–707) shows a 5-Å withdrawal of a phylogenetically invariant glutamine residue from contact with the γ-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F1-type ATP-binding core of the enzyme to an ABC transporter-specific α-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al.(2001)Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the α-helical subdomain as consistent consequences of γ-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel β-type hydrogen bonding interactions between the outermost β-strands in the two core β-sheets, leading to their fusion into a single extended β-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of β-sheet-containing proteins. root mean square adenyl-5′-yl imidodiphosphate ABC (ATP-binding cassette) transporters are ubiquitously distributed ATP-dependent transmembrane solute pumps and ion channels (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (505) Google Scholar, 3Paulsen I.T. Sliwinski M.K. Saier M.H.J. Mol. Biol. 1998; 277: 573-592Crossref Scopus (251) Google Scholar, 4Saurin W. Hofnung M. Dassa E.J. Mol. Evol. 1999; 48: 22-41Crossref PubMed Scopus (252) Google Scholar). These proteins play a causative role in a variety of fatal genetic diseases, including cystic fibrosis (5Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5858) Google Scholar, 6Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar) and adrenoleukodystrophy (7Mosser J. Douar A.M. Sarde C.O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (990) Google Scholar). They also play a role in cancer where overexpression of one family member mediates development of multidrug resistance (2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (505) Google Scholar, 6Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar), a major barrier to effective chemotherapy of advanced malignancies. The fundamental architecture of an ABC transporter comprises a pair of soluble ATP-binding cassettes attached to a pair of α-helical transmembrane domains (each with six to eight transmembrane α-helices). There is unambiguous and strong sequence homology in the ∼200-residue core of the ATP-binding cassettes between even the most remotely related members of the ABC transporter superfamily. However, homology between the transmembrane domains is weak, consistent with their proposed role in determining the diverse transport substrate specificities of the various family members.Previously, the crystal structures of the HisP ATP-binding cassette from Salmonella typhimurium (8Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar) and the MalK ATP-binding cassette from Thermococcus litoralis (9Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (273) Google Scholar) have been reported, establishing the basic fold of the cassette. A crystal structure of a homodimer of the soluble Rad50 DNA repair enzyme has also been reported (10Hopfner K.P. 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 (804) Google Scholar). Although this protein is distantly related to the cassettes from true ABC transporters and lacks the ABC-specific α-helical subdomain, its structure is closely similar to that of HisP in the F1-type (11Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2736) Google Scholar) ATP-binding core and the antiparallel β-subdomain, with the two proteins sharing a 1.11-Å root mean square (r.m.s.)1 deviation for 78 C-α atoms in these regions (compared with an ∼0.40-Å r.m.s. deviation for superposition of the equivalent region in different crystal forms of the same ATP-binding cassette) (see Fig. 1for nomenclature). Based on the fact that the nearly phylogenetically invariant LSGGQ sequence (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 12Schmees G. Stein A. Hunke S. Landmesser H. Schneider E. Eur. J. Biochem. 1999; 266: 420-430Crossref PubMed Scopus (71) Google Scholar) completes the ATPase active site in the 2-fold related cassette in the MgAMP-PNP-bound form of Rad50, this structure established a possible model for the ATP-binding cassette dimer believed to be a conserved feature of all ABC transporters (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (505) Google Scholar, 3Paulsen I.T. Sliwinski M.K. Saier M.H.J. Mol. Biol. 1998; 277: 573-592Crossref Scopus (251) Google Scholar, 4Saurin W. Hofnung M. Dassa E.J. Mol. Evol. 1999; 48: 22-41Crossref PubMed Scopus (252) Google Scholar). Although these crystal structures have provided substantial insight into the structural basis of ABC transporter activity, additional data are needed to define both the structural consequences of the significant sequence variability observed in the N- and C-terminal regions of the cassettes and also the nature of the nucleotide-dependent changes in cassette structure that control the conformational reaction cycle of the ABC transporters. ABC (ATP-binding cassette) transporters are ubiquitously distributed ATP-dependent transmembrane solute pumps and ion channels (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (505) Google Scholar, 3Paulsen I.T. Sliwinski M.K. Saier M.H.J. Mol. Biol. 1998; 277: 573-592Crossref Scopus (251) Google Scholar, 4Saurin W. Hofnung M. Dassa E.J. Mol. Evol. 1999; 48: 22-41Crossref PubMed Scopus (252) Google Scholar). These proteins play a causative role in a variety of fatal genetic diseases, including cystic fibrosis (5Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5858) Google Scholar, 6Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar) and adrenoleukodystrophy (7Mosser J. Douar A.M. Sarde C.O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (990) Google Scholar). They also play a role in cancer where overexpression of one family member mediates development of multidrug resistance (2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (505) Google Scholar, 6Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar), a major barrier to effective chemotherapy of advanced malignancies. The fundamental architecture of an ABC transporter comprises a pair of soluble ATP-binding cassettes attached to a pair of α-helical transmembrane domains (each with six to eight transmembrane α-helices). There is unambiguous and strong sequence homology in the ∼200-residue core of the ATP-binding cassettes between even the most remotely related members of the ABC transporter superfamily. However, homology between the transmembrane domains is weak, consistent with their proposed role in determining the diverse transport substrate specificities of the various family members. Previously, the crystal structures of the HisP ATP-binding cassette from Salmonella typhimurium (8Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar) and the MalK ATP-binding cassette from Thermococcus litoralis (9Diederichs K. Diez J. Greller G. Muller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (273) Google Scholar) have been reported, establishing the basic fold of the cassette. A crystal structure of a homodimer of the soluble Rad50 DNA repair enzyme has also been reported (10Hopfner K.P. 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 (804) Google Scholar). Although this protein is distantly related to the cassettes from true ABC transporters and lacks the ABC-specific α-helical subdomain, its structure is closely similar to that of HisP in the F1-type (11Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2736) Google Scholar) ATP-binding core and the antiparallel β-subdomain, with the two proteins sharing a 1.11-Å root mean square (r.m.s.)1 deviation for 78 C-α atoms in these regions (compared with an ∼0.40-Å r.m.s. deviation for superposition of the equivalent region in different crystal forms of the same ATP-binding cassette) (see Fig. 1for nomenclature). Based on the fact that the nearly phylogenetically invariant LSGGQ sequence (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 12Schmees G. Stein A. Hunke S. Landmesser H. Schneider E. Eur. J. Biochem. 1999; 266: 420-430Crossref PubMed Scopus (71) Google Scholar) completes the ATPase active site in the 2-fold related cassette in the MgAMP-PNP-bound form of Rad50, this structure established a possible model for the ATP-binding cassette dimer believed to be a conserved feature of all ABC transporters (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar, 2Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (505) Google Scholar, 3Paulsen I.T. Sliwinski M.K. Saier M.H.J. Mol. Biol. 1998; 277: 573-592Crossref Scopus (251) Google Scholar, 4Saurin W. Hofnung M. Dassa E.J. Mol. Evol. 1999; 48: 22-41Crossref PubMed Scopus (252) Google Scholar). Although these crystal structures have provided substantial insight into the structural basis of ABC transporter activity, additional data are needed to define both the structural consequences of the significant sequence variability observed in the N- and C-terminal regions of the cassettes and also the nature of the nucleotide-dependent changes in cassette structure that control the conformational reaction cycle of the ABC transporters. We thank Winfried Boos for access to the coordinates of MalK prior to publication as well as Paul Smith and Jacob Keller for assistance with crystallographic data collection. We also acknowledge Wayne Hendrickson for advice and Robert Sweet and Anand Saxena for support during synchrotron data collection at the National Synchrotron Light Source.