The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release.

Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoichiometric antiport without slippage. However, recent NMR studies of the bacterial multidrug transporter EmrE have demonstrated that this multidrug transporter can simultaneously bind drug and proton, which will affect the transport stoichiometry and efficiency of coupled antiport. Here, we investigated the nature of substrate competition in EmrE using multiple methods to measure proton release upon the addition of saturating concentrations of drug as a function of pH. The resulting proton-release profile confirmed simultaneous binding of drug and proton, but suggested that a residue outside EmrE's Glu-14 binding site may release protons upon drug binding. Using NMR-monitored pH titrations, we trace this drug-induced deprotonation event to His-110, EmrE's C-terminal residue. Further NMR experiments disclosed that the C-terminal tail is strongly coupled to EmrE's drug-binding domain. Consideration of our results alongside those from previous studies of EmrE suggests that this conserved tail participates in secondary gating of EmrE-mediated proton/drug transport, occluding the binding pocket of fully protonated EmrE in the absence of drug to prevent dissipative proton transport.

Exploring the potential of boronic acids as inhibitors of OXA-24/40 ß-lactamase.

ß-lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to ß-lactams is the expression of ß-lactamase enzymes. To overcome ß-lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for ß-lactamases also contain the characteristic ß-lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non-ß-lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available ß-lactamase inhibitors, nor are they effectively inhibited by the newest, non-ß-lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C ß-lactamases, and are not extensively characterized as inhibitors of class D ß-lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D ß-lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA-24/40. Several compounds were identified as effective inhibitors of OXA-24/40, with Ki values as low as 5 µM. The X-ray crystal structures of OXA-24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D ß-lactamases.