Studies of the ATP-binding cassette MalK of the maltose ABC transporter MalFGK2 and its inhibitor EIIA
Abstract
ATP-binding cassette (ABC) transporters exist ubiquitously in all organisms from bacteria to humans and actively transport a large variety of solutes across cell membranes. The maltose ABC transporter MalFGK 2 from Escherichia coli consists of two membrane spanning subunits, MalF and MalG, two identical copies of the cytoplasmic ATP-binding cassette, MalK, as well as one periplasmic maltose binding protein, MBP. MalK powers the maltose transporter by binding and hydrolyzing ATP. It is composed of an N-terminal nucleotide binding domain (NBD) and a C-terminal regulatory domain (RD). In the present study, crystal structures of MalK were determined in three dimeric forms: nucleotide free, with ATP bound and with ADP-Mg 2+ bound. The structures show that the regulatory domains of the MalK dimer closely associate, while the nucleotide binding domains open and close like a pair of tweezers depending on whether ATP is present. ATP binding promotes the closure of NBDs, and ATP hydrolysis is required to reset the MalK dimer into the open conformation. The structures of MalK in the resting state (nucleotide free), pre-hydrolysis state (with ATP bound) and post-hydrolysis state (with ADP-Mg2+ bound) shed light on the conformational changes associated with the ATP binding and hydrolysis cycle of ABC transporters. EIIAGlc is a key component of the glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). When glucose is present, EIIA Glc is unphosphorylated, and inhibits certain sugar transporters including MalFGK2 in a process called inducer exclusion. The interaction between EIIAGlc and MalFGK2 was studied using various biochemical and biophysical methods, including a binding assay with fluorescence anisotropy, an inhibition assay, and X-ray crystallography. Zn2+ was found to greatly increase the dissociation constant of MalFGK2 and EIIAGlc. In addition, EIIAGlc binds to MalFGK 2 in the ground state more favorably than in the transition state. This Zn2+-promoted interaction between EIIAGlc and MalFGK2 may be physiologically relevant.
Degree
Ph.D.
Advisors
Chen, Purdue University.
Subject Area
Molecular biology
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