and K.S. stores was inserted into the hydrophobic pit, which is the binding site of the efflux pump inhibitor ABI-PP. LMNG is a substrate of the MexAB-OprM system and competitively inhibits the Cysteamine HCl export of other substrates by this system. However, LMNG does not inhibit the export of other substrates by the inhibitor-binding-pit mutant F178W, which retains the export activity of LMNG. The crystal structure of this mutant suggested that the alkyl chain of LMNG could no longer be inserted into the pit because of steric hindrance. We also determined the crystal structure of MexB containing the high-molecular-mass compound neopentyl glycol derivative C7NG (MW 1,028), the binding site of which overlapped with LMNG in the distal pocket, indicating that whether a substrate binds to the distal or proximal pockets is controlled not only by its molecular weight but also by its individual molecular characteristic. Introduction Antimicrobial resistance (AMR) has been a serious problem in modern chemotherapy since antibiotics were first administered. Currently, the battle between pathogenic bacteria and humanity enters a new stage consisting of the rise and spread of multidrug-resistant (MDR) bacteria amid a decline in the willingness of pharmaceutical companies to develop antibacterial drugs1. Newly emerging MDR gram-negative bacteria over-express RND-type multidrug efflux pumps2,3, which cause resistance against an extraordinarily wide range of antibiotics by a single factor4. RND-type exporters are part of? a tripartite complex, composed of an outer membrane channel, an inner membrane MDR pump and an adaptor protein5. The crystal structures of each component were solved6C8, but the determination of tripartite Cysteamine HCl structures by X-ray crystallography has not succeeded. Instead of the crystal structure, the cryo-EM Cysteamine HCl structures of the complex have been reported9C11. Among the three components of the complex, active energy coupling and substrate recognition are performed by the inner membrane pump12. We first determined the crystal structure of one of the inner membrane MDR pumps, AcrB7. Since then, we have revealed the molecular mechanism of multidrug efflux and multisite recognition by determining the substrate-binding structures of AcrB13C15. MDR pumps are characterized by the difficult in the identification of most bound drugs in their co-crystal structures16. Until now, there are only a few examples of substrate- and inhibitor-bound structures of the physiologically meaningful asymmetrical structures of MDR pumps11,13C15,17C19. For some reason, there are reports of substrate-bound structures of three-fold symmetrical trimers of MDR pumps20C23, while the functionally rotating mechanism of the drug efflux mediated by AcrB14,24C30 is hard to study because of these three-drug-molecule-bound symmetrical trimer structures. If such Cysteamine HCl a symmetrical structure exists with high-molecular-mass compounds. Unexpectedly, these compounds bound to the DBP despite their high molecular mass. Part of their alkyl chains was inserted into the inhibitor-binding pit, and their competitive inhibitory activities to other drug efflux involves the Rabbit Polyclonal to SENP6 insertion of their alkyl chain into the inhibitor-binding pit. Results Crystal structure of MexB bound with Lauryl maltose neopentyl glycol (LMNG) Unlike the main RND transporter from AcrB, bound drugs have not been identified in MexB crystal structures. Regardless of MexB crystalized with or without drugs, the DBP of MexB was occupied by a detergent molecule DDM13,17 (Fig.?1a). In order to identify bound drugs, we exchanged DDM by the large molecular weight detergent LMNG (Fig.?1b), which has a molecular weight almost twice as much as DDM. The molecular weight is not only higher than?that of LMMDs (e.g. minocycline and doxorubicin), but is also higher than that of HMMDs (e.g. rifampicin and erythromycin). We expected that LMNG Cysteamine HCl may not bind to DBP and may not disturb drug binding to the DBP. Open in a separate window Figure 1 Chemical structures of n-dodecyl–D-maltopyranoside (DDM), lauryl maltose neopentyl glycol (LMNG), CYMAL-7 neopentyl glycol (C7NG), and CYMAL-6 neopentyl glycol (C6NG) used in this experiment. Surprisingly, we clearly detected the electron density of bound LMNG in the DBP of the MexB crystal structure, regardless whether or not drug was included in the crystallization medium. Figure?2a,b show the LMNG-bound structure of MexB. The binding site.