Structural And Functional Investigation Of Multidrug Efflux Pump Spr0693-0694-0695 From Streptococcus Pneumoniae

Streptococcus pneumoniae is a Gram-positive bacterium which is responsible for a high burden of human diseases of high death rate.Besides pneumonia,it can also cause other life-threatening diseases such as septicemia,meningitis and sinusitis.Diseases caused by S.pneumoniae are normally treated by antibiotics such as penicillin.To date,several drug-resistant S.pneumoniae isolates has been identified due to the abuse of antibiotics in the past decades.Multidrug resistance pumps are a group of transporters that could extrude many foreign substances out of the cells.Multidrug resistance pumps play an important role in bacterial antibiotic resistance.The Spr0693-0694-0695 system is a multidrug resistance pump which extrude LL-37,a human epithelial antimicrobial peptide.Spr0694-0695 has some features of the ATP-Binding-Cassette(ABC)transporters,in which Spr0694 serves as a nucleotide-binding domain(NBD)that binds and hydrolzcs ATP,while Spr0695 serves as a transmembrane domain(TMD).In this project,using X-ray crystallography,we determined the crystal structures of Spr0693 and Spr0694-0695 to 3.0 A and 3.3 A,respectively.Structural analysis found that Spr0693 is a membrane-fusion protein(MFP),and the hexameric Spr0693 forms a barrel with a substrate exit pore at the center.Spr0694-0695 is a non-canonical ABC transporter,which has a large extracellular domain(ECD)and a transmembrane domain of only eight helices(TM)that possesses no space for substrate entry from the center.Superposition of the structure of Spr0694-0695 to MacAB from Escherichia coli indicated a lateral access tunnel of the substrate.ATPase activity assays in proteoliposomes combined with antimicrobial susceptibility assays enabled us to define a guard helix from the ECD of Spr0695 controls the tunnel,Furthermore.We proposed a putative transport mechanism of this type of multidrug efflax pamp.