Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Dynamics of a Multidrug Resistance Transporter


By: Carol A. Rouzer, VICB Communications
Published: June 25, 2018


Double electron-electron resonance spectroscopy reveals the roles of Na+ and H+ in driving conformational change in a bacterial transporter.    


Multidrug resistance promotes cell survival in the presence of multiple drugs such as antibiotics or cancer chemotherapeutic agents that would normally be toxic. In many cases, multidrug resistance results from the expression of one or more membrane transport proteins that can eject a range of xenobiotic compounds from the cell. There are five families of these transporters of which the multidrug and toxic compound extrusion (MATE) family is an important example. These proteins, found in organisms ranging from bacteria to humans, export mostly positively charged compounds using energy from a Na+ or H+ gradient. Crystal structures are available for five MATE family members. They reveal a common topology comprising 12 transmembrane (TM) helices arranged into separate N-terminal domain (NTD) and C-terminal domain (CTD) bundles. In most cases, the NTD and CTD helical bundles form a V-shaped cavity that is open to the periplasmic (extracellular) side of the membrane, as seen in the crystal structure of the bacterial MATE transporter NorM-Vc from Vibrio cholerae (Figure 1). Because this conformation could favor release of bound substrate to the exterior environment, this structure has been interpreted as an outward-facing state. However, most multidrug transporters are thought to function by transitioning between distinct conformations, called "alternating access", which moves ions and substrates from one side of the membrane to the other. Currently, there are no structures of a MATE family transporter in which the protein occupies an inward facing conformation. Furthermore, data suggest that different family members may use distinct mechanisms for ion-substrate coupling. This led Vanderbilt Institute of Chemical Biology member Hassane Mchaourab and his laboratory to use alternative approaches to investigate the functional dynamics of the NorM-Vc transporter [D. P. Claxton, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online June 18, DOI:10.1073/pnas.1802417115].




FIGURE 1. Ribbon representation of the crystal structure of NorM-Vc. The 12 α-helices are labeled and colored green (1 & 7), red (2 & 8), blue (3 & 9), cyan (4 & 10), dark green (5 & 11), and magenta (6 & 12). (A) View from the plane of the membrane. The NTD domain helices (1-6) and CTD helices (7-12) are easily recognized, as is the large V-shaped cavity opening to the periplasmic side. (B) View from the periplasmic side. (C) View from the cytoplasmic side. Figure generated from PDB #3MTK.



A very effective way to explore the conformational changes that occur during protein function is double electron-electron resonance spectroscopy (DEER), which provides the probability distribution for the distance between two spin labels in the protein under a given set of conditions. Since spin labels are typically introduced site-specifically by modification of cysteine side chains, DEER analysis requires that researchers first construct a mutant protein in which all cysteine residues have been replaced with unreactive residues such as alanine. They then use this cysteine-less protein to design and express a series of mutant proteins, each containing two cysteine residues located at carefully selected sites that are predicted to move with respect to each other during a conformational transition. Once this is accomplished, purified mutant proteins are labeled with a sulfhydryl-targeted spin-label. The Mchaourab lab used the spin label MTSSL [(1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) methanethiosulfonate] for this purpose. After careful verification that the mutagenesis does not significantly affect protein function, the spin-labeled proteins are ready for DEER measurements. For the study of NorM-Vc, the investigators created mutants containing a spin label at position 35 or 45 in TM1 of the NTD and a second spin label in a different NTD helix or a helix in the CTD (Figure 2). These mutants would enable them to explore the effects of binding ions and/or substrate on the movement of TM1 relative to other NTD helices or helices in the CTD. In addition, one mutant contained labels on two different CTD helices to monitor distance changes within the CTD. TM1 contains a highly conserved aspartate residue at position 36 that is known to be critical for transport function. TM1 also forms one side of the V-shaped cavity observed in the crystal structure of NorM-Vc. Thus, the researchers hypothesized that this helix, particularly the region around Asp-36, would be intricately involved in conformational changes during transport, and they designed their labeled mutants to test this hypothesis.




FIGURE 2. Diagrammatic representation of NorM-Vc showing the positions of residues where spin labels were incorporated. Each dotted line connecting two residues (black spheres) indicates the distance between the labels in a mutant transporter that was used for DEER studies. Figure reproduced with permission from D. P. Claxton, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online June 18, DOI:10.1073/pnas.1802417115. Copyright 2018, D. P. Claxton, et al.



An interesting initial observation from the DEER data was that the distances between TM1 and the CTD were smaller and distances between residues within the CTD were larger than expected from the crystal structure of Norm-Vc. This finding suggested that the large V-shaped opening observed in the crystal structure is less favorable when the protein is in solution. The DEER data also revealed that, in the absence of ion or substrate, NorM-Vc exists in multiple conformational states, suggesting flexibility in the protein that could be stabilized by ligand binding. Addition of Na+ ions tended to stabilize conformations with longer distances between the spin labels whereas addition of the substrate doxorubicin (DXR) stabilized conformations with shorter distances. These findings suggested that Na+ and DXR induced different conformations of the protein.  In both cases, distance changes were greater when the labeled residues were located in the two different domains than when they were in different helices of the same domain.

Prior work had shown that an H+ gradient can promote substrate efflux by NorM-Vc. Consistently, DEER data confirmed that H+ ions stabilized conformations of the protein distinct from those stabilized by DXR or Na+. They also used electron paramagnetic resonance spectroscopy to show that H+ and Na+ cause distinct conformational changes – likely bending or rotation – within TM1.

As noted above, Asp-36 is a conserved residue known to be important for NorM-Vc-dependent transport. Other functionally important residues have been identified in a crystal structure of Norm-Vc that includes a Rb+ ion,  an electron-dense substitute for Na+ used for X-ray diffraction studies. The Rb+ ion was found to interact with Glu-255 and Asp-371 in the CTD. As these residues have also been shown to be important for transport, the binding of Rb+ at this site suggested that they may be involved with Na+ binding. In addition, the Mchaourab lab investigators identified a number of highly conserved polar residues (Asn-174, Asn-178, and Thr-200) in the NTD in close proximity to Asp-36 that they hypothesized may play a role in binding and/or transport. To more closely examine the importance of each of these residues, they constructed proteins bearing spin labels at positions 45 in the NTD and 269 in the CTD along with a mutation of one of the residues of interest (Figure 3, 4, and 5). The results of the DEER analysis of these proteins showed that an Asp-36-Asn mutation exhibited no conformational changes in response to Na+ or H+ whereas a Thr-200-Asn mutation responded to H+ but not Na+. Although Na+-driven distance changes were observed in Asn-174-Asp and Asn-178-Asp mutants, the distance between the spin labels was increased in the absence of substrate or ions. Interestingly, introducing a charged side chain via a Thr-200-Lys mutant mimicked an ion-bound conformation. However, the conformational response to DXR binding was not affected by any of these NTD mutations, consistent with the finding that binding affinity for DXR was also unchanged. As a whole, the data suggested that the conserved polar residue network likely forms an ion binding site.



FIGURE 3. (A) Ribbon representation of NorM-Vc as observed in Figure 1A. The circles to the left and right highlight conserved polar residues (colored gold) in the CTD and NTD, respectively, that were investigated in mutation studies. (B) Close-up of the polar residues in the CTD. (C) Close-up of the polar residues in the NTD. Figure generated from PDB #3MTK.




FIGURE 4. Diagrammatic representation of the NTD of NorM-Vc showing the position of the conserved polar residues investigated in mutation studies and the spin label at the site of residue A45. Figure reproduced with permission from D. P. Claxton, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online June 18, DOI:10.1073/pnas.1802417115. Copyright 2018, D. P. Claxton, et al.





FIGURE 5. Diagrammatic representation of the CTD of NorM-Vc showing the position of the conserved polar residues investigated in mutation studies and the spin label at the site of residue L269. Figure reproduced with permission from D. P. Claxton, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online June 18, DOI:10.1073/pnas.1802417115. Copyright 2018, D. P. Claxton, et al.



In contrast with results obtained with NTD mutations, Glu-255-Gln and Asp-371-Asn mutations in the CTD had little effect on the conformational changes induced by Na+ or H+, but they profoundly reduced the structural response to DXR and decreased DXR binding affinity. Since these mutations eliminated the carboxyl group of each amino acid, the mutant proteins lost the potential to acquire a negative charge at these sites through deprotonation. Consistently, binding affinity studies demonstrated a markedly reduced affinity of NorM-Vc for DXR at pH4, conditions under which Glu-255 and Asp-371 would be expected to be fully protonated. These findings suggest that deprotonation of Glu-255 and Asp-371 is required for substrate binding. Furthermore, the finding that mutation of these residues did not affect the conformational response to Na+ does not support the prior hypothesis that they serve as a Na+ binding site.

When the investigators expressed the various mutant NorM-Vc proteins in E. coli, they found that Asp-36-Asn, Glu-255-Gln, Asp-371-Asn, and Thr-200-Lys were substantially impaired in their ability to confer resistance to DXR toxicity. In contrast, Asn-174-Asp, Asn-178-Asp, and Thr-200-Asn exhibited increased activity compared to the corresponding protein bearing the wild-type residue. The poor activity of Asp-36-Asn and Thr-200-Lys was attributed to their reduced ability to respond to ion binding, whereas the poor activity of Glu-255-Gln and Asp-371-Asn was attributed to their reduced ability to bind substrate.

In summary, the data revealed a polar residue network in the NTD of NorM-Vc that mediates conformational changes in response to Na+ and H+ binding. This network had not been previously identified as a Na+ binding site, but the investigators note that these residues are highly conserved, suggesting they may play a role in many transporters. The results also demonstrate the importance of protonation/deprotonation in substrate binding. Together, they suggest that ion entry into the NTD induces conformational transitions that promote substrate release, which is further facilitated by protonation of Glu-255 and Asp-371 in the CTD. Likely, substrate binding is the primary driver for conformational motion in the CTD. These findings offer important new insights into NorM-Vc function that could not be gleaned from the available crystal structure data and provide a foundation for further exploration of the dynamics of the important MATE class of transport proteins.



View PNAS article: Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae








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