title: Interactions and cooperativity between P-glycoprotein structural domains determined by thermal unfolding provides insights into its solution structure and function.
abstract: Structural changes in mouse P-glycoprotein (Pgp) induced by thermal unfolding were studied by differential scanning calorimetry (DSC), circular dichroism and fluorescence spectroscopy to gain insight into the solution conformation(s) of this ABC transporter that may not be apparent from current crystal structures. DSC of reconstituted Pgp showed two thermal unfolding transitions in the absence of MgATP, suggesting that each transition involved the cooperative unfolding of two or more interacting structural domains. A low calorimetric unfolding enthalpy and minimal structural changes were observed, which are hallmarks of the thermal unfolding of α-helical membrane proteins, because generally only the extramembranous regions undergo significant unfolding. Nucleotide binding increased the unfolding temperature of both transitions to the same extent, suggesting that one nucleotide binding domain (NBD) unfolds with each transition. Combined with the results from the two isolated NBDs, we propose that each DSC transition represents the cooperative unfolding of one NBD and the two contacting intracellular loops. Further, the presence of two transitions in both apo and MgATP bound wild-type Pgp suggests the NBD-dimeric conformation is transient, and that Pgp resides predominantly in the crystallographically observed inward-facing conformation with NBDs separated, even under conditions supporting continuous MgATP hydrolysis. In contrast, DSC of the vanadate-trapped MgADP·Pgp complex and the MgATP-bound catalytically inactive mutant, E552A/E1197A, show an additional transition at much higher temperature, corresponding to the unfolding of the nucleotide-trapped NBD-dimeric outward-facing conformation. The collective results indicate a strong preference for an NBD dissociated, inward-facing conformation of Pgp.
authors: Center for Structural Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors: Zhengrong
authors: Z
authors: Yang
authors: Center for Structural Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors: Qingxian
authors: Q
authors: Zhou
authors: Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
authors: Leo
authors: L
authors: Mok
authors: Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
authors: Anukriti
authors: A
authors: Singh
authors: Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
authors: Douglas J
authors: DJ
authors: Swartz
authors: Department of Cell Biology and Biochemistry, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA. Electronic address: ina.urbatsch@ttuhsc.edu.
authors: Ina L
authors: IL
authors: Urbatsch
authors: Center for Structural Biology, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: christie@uab.edu.
authors: Christie G
authors: CG
authors: Brouillette
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013816:Thermodynamics; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011994:Recombinant Proteins; D053835:Unilamellar Liposomes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate
keywords: ABC transporter; Circular dichroism; Detergent; Differential scanning calorimetry; Intrinsic fluorescence; P-glycoprotein