The major facilitator superfamily (MFS) is the largest known superfamily of secondary active transporters. MFS transporters are responsible for transporting a broad spectrum of substrates, either down their concentration gradient or uphill using the energy stored in the electrochemical gradients. Over the last 10 years, more than a hundred different MFS transporter structures covering close to 40 members have provided an atomic framework for piecing together the molecular basis of their transport cycles. Here, we summarize the remarkable promiscuity of MFS members in terms of substrate recognition and proton coupling as well as the intricate gating mechanisms undergone in achieving substrate translocation. We outline studies that show how residues far from the substrate binding site can be just as important for fine-tuning substrate recognition and specificity as those residues directly coordinating the substrate, and how a number of MFS transporters have evolved to form unique complexes with chaperone and signaling functions. Through a deeper mechanistic description of glucose (GLUT) transporters and multidrug resistance (MDR) antiporters, we outline novel refinements to the rocker-switch alternating-access model, such as a latch mechanism for proton-coupled monosaccharide transport. We emphasize that a full understanding of transport requires an elucidation of MFS transporter dynamics, energy landscapes, and the determination of how rate transitions are modulated by lipids.

Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H⁺ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters.

In 2003, a fully automated protein crystallization and monitoring system (PXS) was developed to support the structural genomics projects that were initiated in the early 2000s. In PXS, crystallization plates were automatically set up using the vapor-diffusion method, transferred to incubators and automatically observed according to a pre-set schedule. The captured images of each crystallization drop could be monitored through the internet using a web browser. While the screening throughput of PXS was very high, the demands of users have gradually changed over the ensuing years. To study difficult proteins, it has become important to screen crystallization conditions using small amounts of proteins. Moreover, membrane proteins have become one of the main targets for X-ray crystallography. Therefore, to meet the evolving demands of users, PXS was upgraded to PXS2. In PXS2, the minimum volume of the dispenser is reduced to 0.1 µl to minimize the amount of sample, and the resolution of the captured images is increased to five million pixels in order to observe small crystallization drops in detail. In addition to the 20°C incubators, a 4°C incubator was installed in PXS2 because crystallization results may vary with temperature. To support membrane-protein crystallization, PXS2 includes a procedure for the bicelle method. In addition, the system supports a lipidic cubic phase (LCP) method that uses a film sandwich plate and that was specifically designed for PXS2. These improvements expand the applicability of PXS2, reducing the bottleneck of X-ray protein crystallography.

Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria.

Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. Here we describe an approach to create artificial allosteric sites in protein complexes. Certain protein complexes contain subunits with pseudo-active sites, which are believed to have lost functions during evolution. Our hypothesis is that allosteric sites in such protein complexes can be created by restoring the lost functions of pseudo-active sites. We used computational design to restore the lost ATP-binding ability of the pseudo-active site in the B subunit of a rotary molecular motor, V₁-ATPase. Single-molecule experiments with X-ray crystallography analyses revealed that binding of ATP to the designed allosteric site boosts this V₁’s activity compared with the wild-type, and the rotation rate can be tuned by modulating ATP’s binding affinity. Pseudo-active sites are widespread in nature, and our approach shows promise as a means of programming allosteric control over concerted functions of protein complexes.