Crystal Structure of the Open State of the Neisseria gonorrhoeae MtrE Outer Membrane Channel

Abstract

Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel.

Crystallization of Membrane Proteins by Vapor Diffusion

“…8b). 51 Analysis of the Matthews coefficient indicated the presence of one MtrE protomer (49.29 kDa) per asymmetric unit, with a solvent content of 75.8%. The final structural model was resolved to a resolution of 3.3 Å.…”

“…The most common and successful method of membrane protein crystallization, to date, this approach has allowed researchers in our lab to determine the crystal structures of a number of membrane proteins. These include the inner membrane efflux pumps CusA, 47 AcrB, 48 and MtrD, 49 the outer membrane channels CusC, 50 MtrE, 51 and CmeC, 52 as well as the CusBA adaptor-transporter efflux complex. 53 …”

Assembly and operation of bacterial tripartite multidrug efflux pumps

“…Clearly this constriction must somehow open at some stage of the transport process. Insight into the channel opening process has been provided from crystal structures of partially opened states of TolC [26,27] and the homologous Neisseria gonorrhoeae MtrE (a component of the MtrC-MtrD-MtrE tripartite pump) [28]. These structures show that the opening is associated with a change in the superhelical trajectory of the coiled coils and with prying apart of the gating salt bridges that stabilize the closed state.…”

Neisseria gonorrhoeae Defense against Human Neutrophils and Host-derived Antimicrobials

“…Neutrophils store high concentrations of the LL-37 precursor, hCAP18, in secondary granules (Wiesner and Vilcinskas, 2010), which could explain the importance of MtrCDE for Gc resistance to the degranulated supernatant released from activated neutrophils. However, neutrophils also possess other antimicrobials with amphipathic characteristics including α-defensins (HNPs 1-4), lysozyme, bactericidal/permeability-increasing protein (BPI), CAP37/azurocidin, lactoferrin and cathepsin G. The solved structure of the MtrE outer membrane channel revealed an exit pore of approximately 22 Å in diameter (Lei et al, 2014), which would exclude higher molecular weight proteins such as BPI (55kDa), CAP37 (37kDa) and lactoferrin (80kDa), but could feasibly facilitate export of HNPs (~4kDa), lysozyme (14.5kDa) and cathepsin G (26kDa). In addition, some of the larger neutrophil proteins including lactoferrin are proteolytically processed by host proteases to release active antimicrobial peptide fragments (Wiesner and Vilcinskas, 2010;Sinha et al, 2013), which are within the size range for export by MtrCDE.…”

“…The three components form a functional pump that spans the inner and outer membrane of Gram-negative bacteria, and exports harmful compounds completely out of the cell. MtrD is a proton motive force-dependent, inner-membrane channel, which connects to the MtrE outer-membrane channel through the MtrC membrane-fusion protein and assembles in a 3:6:3 stoichiometry (MtrD:MtrC:MtrE) (Lei et al, 2014). Expression of MtrCDE is controlled by both a transcriptional repressor (MtrR) and an activator (MtrA) (Zalucki et al, 2012).…”