Interdomain communication in a homodimeric ABC transporter

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• ABC transporters facilitate solute transport across cellular membranes, powered by ATP.
• Dysfunctional ABC transporters are linked to diseases like cystic fibrosis and antibiotic resistance.
• Interdomain communication in type IV ABC transporters remains poorly understood.

Purpose of the Study:

• To elucidate the function of conserved charged residues in the intracytosolic loop 1 of the human lysosomal peptide transporter TAPL.
• To investigate the role of specific residues in mediating interdomain communication and regulating transport activity.

Main Methods:

• Site-directed mutagenesis of three conserved charged residues (D278, R288, D292) in TAPL.
• Assays to measure peptide transport and ATPase activity of wild-type and mutant TAPL transporters.
• Analysis of nucleotide specificity (ATP vs. GTP) for hydrolysis and transport.

Main Results:

• D278A substitution disrupted peptide transport by inhibiting ATP hydrolysis.
• R288A and D292A substitutions reduced peptide transport but increased basal ATPase activity.
• R288A and D292A mutants showed altered peptide-dependent ATPase activity and hydrolyzed both ATP and GTP, unlike wild-type TAPL.

Conclusions:

• D278 is vital for bidirectional interdomain communication via polar interactions.
• R288 and D292 regulate ATP hydrolysis, likely by stabilizing the outward-facing transporter conformation.
• Understanding these residues' functions advances knowledge of ABC transporter mechanisms and potential therapeutic targets.