Nucleotide State Regulates Asymmetric Substrate-Binding Protein Docking in a Type II ABC Importer

Abstract

Type II ABC importers rely on substrate-binding proteins (SBP) to capture nutrients and deliver them to membrane-embedded transporters, but how ATP binding and hydrolysis regulate their interactions remains poorly understood. To address these questions, we determined structures of the Type II small-substrate molybdate importer MolBC-A from H. influenzae in distinct nucleotide-bound states, complemented by molecular dynamics (MD) simulations and functional assays. In a nucleotide-free state, the MolA-MolBC interface is remodeled into a more open conformation, whereas ATP and AMP-PNP binding induce asymmetric conformational changes that preserve the MolA Lobe I interface and disrupt contacts with MolA Lobe II, revealing a previously unresolved partially-docked state. In the ADP-bound state, MolA exhibits the greatest conformational heterogeneity with reduced stable engagement that extends across both lobes. Together these findings define a nucleotide-dependent cycle of asymmetric MolA remodeling and reveal distinct contributions of the two SBP lobes in coupling transporter nucleotide state to SBP engagement.