Distinct structural and functional properties of the ATPase sites in an asymmetric ABC transporter.

The ABC transporter associated with antigen processing (TAP) shuttles cytosolic peptides into the endoplasmic reticulum for loading onto class I MHC molecules. Transport is fueled by ATP binding and hydrolysis at two distinct cytosolic ATPase sites. One site comprises consensus motifs shared among most ABC transporters, while the second has substituted, degenerate motifs. Biochemical and crystallography experiments with a TAP cytosolic domain demonstrate that the consensus ATPase site has high catalytic activity and facilitates ATP-dependent dimerization of the cytosolic domains, which is an important conformational change during transport. In contrast, the degenerate site is defective in dimerization and ATP hydrolysis. Full-length TAP mutagenesis demonstrates the necessity for at least one consensus site, supporting our conclusion that the consensus site is the principal facilitator of substrate transport. Since asymmetry of the ATPase site motifs is a feature of many mammalian homologs, our proposed model has broad implications for ABC transporters.

[1]  Li-Wei Hung,et al.  Crystal structure of the ATP-binding subunit of an ABC transporter , 1998, Nature.

[2]  S. Cole,et al.  Comparison of the Functional Characteristics of the Nucleotide Binding Domains of Multidrug Resistance Protein 1* , 2000, The Journal of Biological Chemistry.

[3]  J. Bluestone,et al.  Substitution at residue 227 of H–2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes , 1989, Nature.

[4]  R. Tampé,et al.  Peptides Induce ATP Hydrolysis at Both Subunits of the Transporter Associated with Antigen Processing* , 2003, Journal of Biological Chemistry.

[5]  John F Hunt,et al.  ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer. , 2002, Molecular cell.

[6]  R. Tampé,et al.  Functional Non-equivalence of ATP-binding Cassette Signature Motifs in the Transporter Associated with Antigen Processing (TAP)* , 2004, Journal of Biological Chemistry.

[7]  A. E. Senior,et al.  The catalytic cycle of P‐glycoprotein , 1995, FEBS letters.

[8]  O. Daumke,et al.  Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle , 2001, Current Biology.

[9]  P. Lehner,et al.  The ABC‐transporter signature motif is required for peptide translocation but not peptide binding by TAP , 2003, European journal of immunology.

[10]  A. Townsend,et al.  Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter , 1991, Nature.

[11]  Rachelle Gaudet,et al.  Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing , 2001, The EMBO journal.

[12]  Douglas C. Rees,et al.  The E. coli BtuCD Structure: A Framework for ABC Transporter Architecture and Mechanism , 2002, Science.

[13]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[14]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[15]  R. Tampé,et al.  Functional Dissection of the Transmembrane Domains of the Transporter Associated with Antigen Processing (TAP)* , 2004, Journal of Biological Chemistry.

[16]  John F Hunt,et al.  Cooperative, ATP-dependent Association of the Nucleotide Binding Cassettes during the Catalytic Cycle of ATP-binding Cassette Transporters* , 2002, The Journal of Biological Chemistry.

[17]  A. M. George,et al.  The ABC transporter structure and mechanism: perspectives on recent research , 2004, Cellular and Molecular Life Sciences CMLS.

[18]  D. Herschlag,et al.  Mapping the transition state for ATP hydrolysis: implications for enzymatic catalysis. , 1995, Chemistry & biology.

[19]  M. Sansom,et al.  Nucleotide binding to the homodimeric MJ0796 protein: A computational study of a prokaryotic ABC transporter NBD dimer , 2005, FEBS letters.

[20]  R. Neubig,et al.  Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding* , 2001, The Journal of Biological Chemistry.

[21]  K. Linton,et al.  The ATP switch model for ABC transporters , 2004, Nature Structural &Molecular Biology.

[22]  P. Wyatt.  Light scattering and the absolute characterization of macromolecules , 1993 .

[23]  P. Cresswell,et al.  Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP) , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Robert DeMars,et al.  Restored expression of major histocompatibility class I molecules by gene transfer of a putative peptide transporter , 1991, Nature.

[25]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[26]  P. Cresswell,et al.  Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP. , 1996, Immunity.

[27]  P. Parham,et al.  Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8 , 1989, Nature.

[28]  P. van Endert,et al.  Tapasin Enhances Assembly of Transporters Associated with Antigen Processing-dependent and -independent Peptides with HLA-A2 and HLA-B27 Expressed in Insect Cells* , 1999, The Journal of Biological Chemistry.

[29]  B. Dijkstra,et al.  Formation of the productive ATP-Mg2+-bound dimer of GlcV, an ABC-ATPase from Sulfolobus solfataricus. , 2003, Journal of molecular biology.

[30]  R. Tampé,et al.  A sequential model for peptide binding and transport by the transporters associated with antigen processing. , 1994, Immunity.

[31]  A. M. George,et al.  Subunit interactions in ABC transporters: towards a functional architecture. , 1999, FEMS microbiology letters.

[32]  A Vagin,et al.  An approach to multi-copy search in molecular replacement. , 2000, Acta crystallographica. Section D, Biological crystallography.

[33]  Rachelle Gaudet,et al.  Structure of the N-terminal Ankyrin Repeat Domain of the TRPV2 Ion Channel* , 2006, Journal of Biological Chemistry.

[34]  F. Quiocho,et al.  A tweezers-like motion of the ATP-binding cassette dimer in an ABC transport cycle. , 2003, Molecular cell.

[35]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[36]  D. Herschlag,et al.  Ras-catalyzed hydrolysis of GTP: a new perspective from model studies. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[37]  G. Chang,et al.  Structure of the ABC Transporter MsbA in Complex with ADP·Vanadate and Lipopolysaccharide , 2005, Science.

[38]  S. Cole,et al.  Role of Carboxylate Residues Adjacent to the Conserved Core Walker B Motifs in the Catalytic Cycle of Multidrug Resistance Protein 1 (ABCC1)* , 2003, Journal of Biological Chemistry.

[39]  T. Elliott,et al.  Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex , 1992, Nature.

[40]  P. Cresswell,et al.  Human transporters associated with antigen processing possess a promiscuous peptide-binding site. , 1994, Immunity.

[41]  J. Hunt,et al.  Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter. , 2001, Structure.

[42]  Lutz Schmitt,et al.  H662 is the linchpin of ATP hydrolysis in the nucleotide‐binding domain of the ABC transporter HlyB , 2005, The EMBO journal.

[43]  H. Ploegh,et al.  HLA-A- and HLA-B-specific monoclonal antibodies reactive with free heavy chains in western blots, in formalin-fixed, paraffin-embedded tissue sections and in cryo-immuno-electron microscopy. , 1990, International immunology.

[44]  John A. Tainer,et al.  Structural Biology of Rad50 ATPase ATP-Driven Conformational Control in DNA Double-Strand Break Repair and the ABC-ATPase Superfamily , 2000, Cell.

[45]  P. Lapinski,et al.  Nucleotide Interactions with Membrane-bound Transporter Associated with Antigen Processing Proteins* , 2003, The Journal of Biological Chemistry.

[46]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[47]  D. Wiley,et al.  Identification of domain boundaries within the N‐termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin‐mediated increase in peptide loading of MHC class I , 2005, Immunology and cell biology.

[48]  P. van Endert,et al.  Powering the peptide pump: TAP crosstalk with energetic nucleotides. , 2002, Trends in biochemical sciences.