Unveiling the structural details of the nucleotide-binding protein of an antimicrobial peptide transporter from non-typeable Haemophilus influenzae.

Abstract

Non-typeable Haemophilus influenzae (NTHi) is an opportunistic extracellular pathogen associated with various respiratory diseases. To combat host antimicrobial peptides, NTHi has developed a specialized transporter named Sap (sensitivity to antimicrobial peptide). Within this transporter, the HiSapF component functions as a nucleotide-binding domain (NBD). Despite its significant role, the structural and functional characteristics of HiSapF remained unexplored. In this study, we present the crystal structure of HiSapF (2.3 Å resolution) from NTHi, which features nine α-helices and nine β-sheets, including three conserved antiparallel β-sheets (β1-β2-β3) in the N-terminal region. Key structural features (Walker A and B motifs, Q loop, D loop, and signature motifs) are conserved in HiSapF, aligning with known NBDs. Computational and biophysical analyses indicated that HiSapF can possibly self-associate to dimerize under the tested conditions. Further comparative molecular docking, dynamic simulations, and mutational studies reveal that the residues from Walker A and B motifs of HiSapF are essential for interacting with ATPMg2+. Specifically, the Walker A residues primarily engage the adenosine group, while the Walker B residues (conserved residue Asp173), identified as crucial for coordinating the Mg2+ ion of the ATP molecule for hydrolysis (Km: ~2.34 ± 0.32 mM; kcat: ~0.025 s-1). Novobiocin, identified as a potential inhibitor of HiSapF, demonstrates a putative allosteric-binding site and can reduce ATPase activity; further structural and biochemical studies can enhance our understanding. Collectively, these findings offer important structural and functional insights into HiSapF and may facilitate further investigation for the development of inhibitors targeting NBDs against bacterial transporters.