Prof. Shimon Schuldiner
Recent news about some of my favorite proteins
On the link between antibiotic resistance, diabetes, and wastewater
These seemingly unrelated issues of antibiotic resistance, diabetes, and wastewater are actually interconnected. Grasping this connection is essential in addressing the challenges we face today and preventing future ones.
A recent study conducted by Stockbridge's laboratory has found a link between Small Multidrug Resistance (SMR) transporters and the metabolism of metformin, a common drug used to treat type II diabetes. Previous research shows that microbial communities play a crucial role in the breakdown of metformin in the environment, and SMR transporters, particularly SMRGdx, are believed to be necessary for this process. Metformin, a biguanide drug, is structurally similar to nitrogenous waste products such as guanidinium (Gdm+) and guanylurea, which SMRGdx exports. The study conducted by Lucero et al. (Lucero et al., 2024) used various methods to investigate how SMRGdx and other SMR proteins interact with metformin metabolites. These proteins are linked to horizontal gene transfer in wastewater bacteria that degrade metformin.
The research provides a comprehensive analysis of the SMRGdx subtype, including biochemical, structural, and mechanistic attributes. These findings are relevant not only to other members of the SMR family but also to other multidrug transporters (MDTs), proteins best known for their role in microbial antibiotic resistance. (this issue: (Lucero et al., 2024)).
II. Metformin in wastewater
Metformin is a widely prescribed drug for type II diabetes. It is taken in gram quantities daily by over 150 million people and excreted in an unchanged form, along with its degradation product guanylurea (Foretz et al., 2023). These two compounds are the most frequently found anthropogenic chemicals in wastewater worldwide, with concentrations measured up to the low µM range in sampled waste and surface waters. Unfortunately, typical wastewater treatment protocols do not remove these compounds, leading to environmental concerns about their accumulation in surface water (Briones et al., 2016).
Interestingly, metformin has been linked to changes in the composition of microbial communities in the gut and wastewater treatment plants. Some studies suggest that it may act as a co-selective agent, promoting the survival of antibiotic-resistant bacteria in the presence of antibiotics. On the other hand, recent research has identified bacteria that use metformin as a nitrogen and carbon source, indicating that biodegradation of metformin and guanylurea could be a viable strategy for remediating these compounds (Martinez-Vaz et al., 2022).
III. Multidrug Transporters (MDTs) and Antibiotic Resistance
Multidrug transporters (MDTs) are membrane proteins that recognize a wide range of antibiotics. They remove the antibiotics from the cell in an energy-dependent process and are responsible for the resistance in some microorganisms. A prominent feature of these Multidrug Transporters (MDTs) is their extremely broad specificity, which means that a single transporter can confer resistance against various drugs. Some MDTs have even been reported to confer resistance to dozens of toxic compounds with few common features. A hypothesis suggests that each multidrug transporter has evolved to remove a specific natural compound or a group of similar ones. This means that they are not substantially different from regular transporters. The ability of multidrug transporters to remove toxins is considered an opportunistic side effect that is only detected when bacteria are exposed to drugs in experimental or clinical situations. The concept that MDTs may have other natural functions was first suggested in B. subtilis, where it was shown that the transporter Blt is part of an operon that detoxifies spermidine (Neyfakh, 1997). Many other MDTs have since been associated with various biological functions, including removing, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals, and bacterial metabolites such as breakdown products of metformin. Furthermore, mammalian homologs have evolved to fulfill various functions, including neurotransmitter transport, reabsorption of essential molecules, or secretion of toxic ones in the kidney (Henderson et al., 2021, Schuldiner et al., 1995).
IV. The SMR Family and Metformin Transport
The SMR family comprises small four-transmembrane helical proteins known for their role in antibiotic and antiseptic resistance (Schuldiner, 2009, Burata et al., 2022). These proteins are among the smallest membrane transport proteins, perfect candidates for detailed biochemical and biophysical analysis. EmrE, has been the prototype for members of the SMR family and is the most extensively studied member of the SMRQac subtype, It is known to transport substrate across the cell membrane by exchanging protons. EmrE possesses a unique characteristic - it has only one membrane-embedded charged residue, Glu14, which is conserved in hundreds of homologous proteins in bacteria and archaea. Glu14 plays a crucial role in the coupling mechanism, as its deprotonation is necessary for substrate binding (Schuldiner, 2009).
There are two types of SMR, each with different abilities to transport substances. Stockbridge et al. previously showed that while SMRQac can transport various antimicrobial substances, including antiseptics like benzalkonium, which are commonly found in wastewater, SMRGdx is highly specific and do not provide robust resistance to classical antimicrobials. In its primary physiological context, SMRGdx exports the nitrogenous waste product Gdm+ and its breakdown product guanylurea (Burata et al., 2022). The aforementioned evidence supports a role of SMRGdx in the metabolism and biodegradation of Metformin. Therefore, the study in this issue embarks on a detailed analysis of the molecular and evolutionary implications of this finding.
In this study, the Stockbridge lab investigated whether several genomic and plasmid-associated SMRs transport metformin or other byproducts of microbial metformin metabolism. They examined four SMRGdx homologs, including Gdx-Clo, the structurally characterized genomic protein from Clostridales (Kermani et al., 2020).
The study revealed that genomic and plasmid-associated SMRGdx homologs efficiently transport guanylurea with transport kinetics similar to the physiological substrate Gdm+ . In an elegant part of the study, metformin metabolites were tested for transport using solid-supported membrane (SSM) electrophysiology. The experiment involved reconstituting proteoliposomes with purified proteins to monitor charge movement across the liposome membrane. The Gdx-Clo homolog, the best characterized SMRGdx homolog, was found to transport only Gdm+ and guanylate. However, the other three SMRGdx homologs tested also transported singly-substituted biguanides, including the metformin degradation product methyl biguanide and the related antidiabetic drug buformin. On the other hand, metformin, a doubly substituted biguanide, exhibited currents barely above the detectable limit by SMRGdx proteins. These findings are consistent with prior observations that SMRGdx transports guanidinium ions with single hydrophobic substitutions, while doubly-substituted guanidiniums are not.
Previous research studies have shown that both Gdx-Eco and EmrE have a well-coupled 2 H+: 1 Gdm+ stoichiometry. However, it has been suggested that the transport stoichiometry may vary among some transported substances for EmrE. To determine the coupling stoichiometry of Gdx-Clo and plasmid-associated Gdx-pAmi, Stockbridge et al. conducted experiments using the SSM setup. In these experiments, a potassium gradient and the potassium ionophore valinomycin were used to establish several membrane potentials, and pyranine, a pH-sensitive fluorescent dye, was used to monitor substrate-coupled proton movement. When the equilibrium reversal potential was established, no substrate movement was detected. The equilibrium potential detected, which was -60 mV in this case, is in agreement with a 2 H+: 1 solute coupling stoichiometry.
Lucero et al. solved a crystal structure of Gdx-Clo in the presence of guanylurea to investigate if it occupies the same binding site as guanidinium in Gdx-Clo. The crystal structure of Gdx-Clo with guanylurea was solved up to 2.1 Å, which showed that guanylurea occupies the same binding pocket as Gdm+ in Gdx-Clo. The Gdm+group is positioned between the central glutamates.
This research holds significant implications for the scientific community. It not only offers a comprehensive examination of the structurally characterized SMRGdx homolog Gdx-Clo, laying a solid groundwork for future mechanistic studies of this model transport protein, but also shines a spotlight on the potential role of SMRGdx transporters in the microbial management of metformin and its microbial metabolic byproducts. These findings reveal how native transport physiologies adapt to new selective pressures, underlining the relevance of the reported findings to microbiology and drug resistance.
Briones, R. M., Sarmah, A. K. & Padhye, L. P. 2016. A global perspective on the use, occurrence, fate and effects of anti-diabetic drug metformin in natural and engineered ecosystems. Environ Pollut, 219, 1007-1020.
Burata, O. E., Yeh, T. J., Macdonald, C. B. & Stockbridge, R. B. 2022. Still rocking in the structural era: a molecular overview of the Small Multidrug Resistance (SMR) transporter family. Journal of Biological Chemistry, 298, 102482.
Foretz, M., Guigas, B. & Viollet, B. 2023. Metformin: update on mechanisms of action and repurposing potential. Nat Rev Endocrinol, 19, 460-476.
Henderson, P. J. F., Maher, C., Elbourne, L. D. H., Eijkelkamp, B. A., Paulsen, I. T. & Hassan, K. A. 2021. Physiological Functions of Bacterial "Multidrug" Efflux Pumps. Chem Rev, 121, 5417-5478.
Kermani, A. A., Macdonald, C. B., Burata, O. E., Ben Koff, B., Koide, A., Denbaum, E., Koide, S. & Stockbridge, R. B. 2020. The structural basis of promiscuity in small multidrug resistance transporters. Nature Communications, 11, 6064.
Lucero, R. M., Demirer, K., Yeh, T. J. & Stockbridge, R. B. 2024. Transport of metformin metabolites by guanidinium exporters of the Small Multidrug Resistance family. this issue
Martinez-Vaz, B. M., Dodge, A. G., Lucero, R. M., Stockbridge, R. B., Robinson, A. A., Tassoulas, L. J. & Wackett, L. P. 2022. Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products. Front Bioeng Biotechnol, 10, 1086261.
Neyfakh, A. 1997. Natural functions of bacterial multidrug transporters. Trends Microbiol. , 5, 309-313.
Schuldiner, S. 2009. EmrE, a model for studying evolution and mechanism of ion-coupled transporters. Biochim Biophys Acta, 1794, 748-762.
Schuldiner, S., Shirvan, A. & Linial, M. 1995. Vesicular neurotransmitter transporters: from bacteria to humans. Physiol Rev, 75, 369-92.
VMAT structures reveal exciting targets for drug development
Shimon Schuldiner* and Lucy R. Forrest*
The Vesicular Monoamine Transporter 2 (VMAT2) plays a crucial role in neurotransmission of biogenic amines. Recently, four research teams, led by Coleman, Zhang and Lee, Zhao and Jiang, and Wang and Qu, reported Cryo-EM structures of human VMAT2, offering opportunities for developing improved therapeutics and deep insights into the functioning of this protein.
Vesicular monoamine transporters (VMATs) actively remove monoamines such as serotonin, dopamine, histamine, adrenaline, and noradrenaline from the cytosol. These monoamines are thus stored in synaptic vesicles at nerve terminals or storage organelles found in platelets, enterochromaffin, chromaffin, and other similar cells. This storage process is essential in regulating the release of neurotransmitters into the synaptic cleft or to appropriate targets throughout the human body [1, 2].
In humans, mutations in VMATs can lead to developmental delay, dystonia, parkinsonism, and increased mortality, highlighting the importance of these transporters in neurotransmission. VMATs are also a target for treating hypertension, psychotic agitation, psychostimulant abuse, and hyperkinetic movement disorders such as Huntington's disease-related chorea [2, 3].
Despite their physiological and pharmacological significance, the structural basis for substrate recognition and inhibition by different drugs was unknown until recently. Four publications reported Cryo-EM structures of VMAT2 in different conformations, bound to substrates and inhibitors. Here we explain the important implications in understanding the protein's workings and in developing more potent and clinically relevant inhibitors.
II. Drugs targeting VMAT
There are two isoforms of vesicular monoamine transporters (VMATs): VMAT1 and VMAT2. Although they share high sequence similarity, their tissue distributions and sensitivity to inhibitors differ. While there may be some differences in the expression of VMAT isoforms depending on the species, VMAT2 is mainly found in neurons in humans, while VMAT1 is present in neuroendocrine cells.
Reserpine is an indole alkaloid that has been used in the treatment of antipsychotic and antihypertensive disorders. However, it is now considered a second-line therapy because of its potential side effects. It works by competitively inhibiting both VMAT isoforms. Tetrabenazine (TBZ), on the other hand, is a noncompetitive inhibitor of only VMAT2. It is used to treat hyperkinetic disorders associated with Huntington's disease and Tourette's syndrome. TBZ derivatives have also been used in studies of neurodegenerative and psychiatric diseases through in vivo positron emission tomography (PET) imaging of VMAT2 density.
Ketanserin (KET), an antagonist of the 5-HT2 receptor, is an antihypertensive agent that potently inhibits VMAT non-competitively. In addition to biogenic amines, VMAT can also transport non-natural substrates such as the neurotoxin N-methyl-4-phenyl pyridinium (MPP), psychostimulant amphetamines, and several other toxicants.
III. Transport mechanism of monoamines via VMATs
The transport of monoamines via VMATs is coupled with the movement of two protons in opposite directions and depends on the proton electrochemical gradient generated by the vesicular H+-ATPase [1, 4].
For more than 50 years, the working model of substrate transport has been the alternating access mechanism. According to this mechanism, a conformational change exposes one binding site alternately to either side of the membrane . In the case of an antiporter like VMAT, the conformational change occurs only when the substrate or one or more protons are bound, not when the binding site is empty [4, 5].
IV. Recent studies on VMAT2 structures and interactions with substrates and inhibitors
Four recent publications report structures of human VMAT2 solved with cryo-EM [6-9]. As observed in all major facilitator superfamily (MFS) structures to date, the VMAT2 structures comprise two lobes of six membrane-spanning helices. These core regions are relatively small and lack features required for cryo-EM image processing, a problem that three of the teams have solved by appending two interacting protein domains to short loops connecting to the first and last transmembrane helices. All four studies reported structures in the presence of TBZ, whose pocket is capped on the lumenal side in large part by the lumenal end of transmembrane (TM) helices 1 and 7, which adopts a distinct conformation (Fig. 1, orange). As may have been expected from pharmacological and biochemical studies, the transporter is also fully occluded on the cytosolic side when bound to TBZ. The four studies agree on the location of the TBZ ligand, and they demonstrate the importance of identified interactions using mutagenesis. The details provided by these structures reveal the interactions conferring the differences in specificity between VMAT1 and VMAT2. Accordingly, the studies provide a promising foundation for development of drugs with similar noncompetitive profile to TBZ. Calculations of the pKa of the TBZ-bound structures with empirical methods suggest that TBZ is deprotonated and is accompanied by two protons in the binding site [6, 9], although molecular dynamics simulations of those states gave a more mixed picture. Thus, higher-level computations, such as quantum chemical or free energy perturbations, will be of value to establish the role of protons in high affinity binding by TBZ-like compounds.
Relevant to proton binding, the new structures also confirm the locations for four acidic residues previously identified as critical: D33, E312, D399, and D426 (Fig. 1). While two of them, E312 and D399, directly interact with serotonin, D33 and D426 are involved in a broad hydrogen bond network spanning both the N- and C-terminal lobes and are the likely proton carriers. It remains to be established how binding of the two protons facilitates the required conformational change, but it is worth noting that D33 is highly conserved across MFS transporters.
As mentioned above, binding of TBZ is known to occur by a noncompetitive mechanism. A likely origin of this noncompetitive nature is that it traps the transporter in an occluded conformation inaccessible to substrates. Reserpine, a competitive inhibitor, by contrast, requires the proton gradient to bind. This feature presents a challenge when handling purified protein. Therefore, to obtain a reserpine-bound state, Wu et al. purified the protein after its exposure to Reserpine while Pidathala et al. and Wang et al. used cytoplasmic gating residue mutants, Y418S or Y422C, which allow VMAT2 to adopt the cytosolic-open conformation in the absence of a gradient. The structure of reserpine bound to VMAT2 revealed a wide-open pathway, with the ligand binding site closer to the cytoplasmic side (Fig. 1). Reserpine occupies the pathway in a way that suggests that it would compete with substrate binding competitively. Notably, the reserpine and TBZ sites overlap only minimally, providing two entirely different frameworks for drug development.
Beyond inhibitor binding, a comparison of the outward and inward-facing structures allowed examining the key elements of the transport-associated conformational change, which involve relative translations of the N- and C-terminal lobes. As for the other secondary active transporter families, such as the neurotransmitter sodium symporters and the excitatory amino acid transporters, the underlying alternating access mechanism in the MFS family arises from exchanging conformations of inverted-topology repeats .
It is important to consider, however, that these structures may be distorted relative to substrate-bound states due to the presence of the inhibitors. Three studies addressed this by also determining structures in the presence of serotonin, either in the wild type [8, 9]or cytosolic-open mutants . Of the reported 5-HT-bound structures, the higher-resolution structure is of wild-type VMAT2 in a lumen-facing state and suggests a binding site in which the indole ring of serotonin overlaps with the larger indole ring system of reserpine . Both ligands interact with E312 (Fig. 1), a residue previously identified to be critical based on similarity to bacterial transporters. However, simulations combined with continuum electrostatics calculations also implicate D399 as a key site for protonation during the transport cycle .
Taken together, the structural studies validate a paradigm shift in development of VMAT2 inhibitors. Indeed, while AlphaFold models have improved the quality of VMAT2 predictions over homology-based approaches, they differ from the structural data in interactions predicted in key regions, such as the occlusion of the lumenal side.
The conformation-dependent binding of inhibitors and the broad specificity that enables VMAT to attach to a wide range of molecules make it an exciting target for further study and potential therapeutic development.
1 Schuldiner, S., et al. (1995) Vesicular neurotransmitter transporters: from bacteria to humans. Physiol Rev75, 369-392
2 Blakely, R.D. and Edwards, R.H. (2012) Vesicular and plasma membrane transporters for neurotransmitters. Cold Spring Harb Perspect Biol 2012;4:a005595
3 Alwindi, M. and Bizanti, A. (2023) Vesicular monoamine transporter (VMAT) regional expression and roles in pathological conditions. Heliyon 9, e22413
4 Yaffe, D., et al. (2018) The ins and outs of vesicular monoamine transporters. J Gen Physiol 150, 671-682
5 Drew, D., et al. (2021) Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS). Chemical reviews 121, 5289-5335
6 Dalton, M.P., et al. (2023) Structural Mechanisms for VMAT2 inhibition by tetrabenazine. eLife. https://doi.org/10.7554/eLife.91973.1
7 Pidathala, S., et al. (2023) Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2. Nature 623, 1086-1092
8 Wu, D., et al. (2023) Transport and inhibition mechanisms of human VMAT2. Nature https//doi.org/ 10.1038/s41586-023-06926-4.
9 Wang, Y., et al. (2024) Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines. Cell research 34, 47-57
10 Forrest, L.R. (2013) Structural biology. (Pseudo-)symmetrical transport. Science 339, 399-401
Figure 1: Structures of human VMAT2 reveal different inhibitor binding profiles. Structures have been determined in the presence of three inhibitors: (A) reserpine, (B) tetrabenazine, (C) ketanserin; and a substrate serotonin (D), shown as sticks. Key acidic residues are also shown as sticks (see text). The structures reflect distinct conformational states: open to the cytoplasm (A), completely occluded (B), and open to the lumenal side (C, D). Pathway opening involves relative "rocker switch" movements of the N- and C-terminal halves of the transporter. The pathways on either side of the transporter involve membrane-spanning helices from pseudo-symmetric repeated elements, highlighted by the positions of the first helix of each repeat (TM 1 in green, TM4 in blue, TM7 in orange and TM10 yellow). Figures created with PyMol for PDB identifiers 8T6A, 8T69, 8JT9 and 8JSW, respectively, and compiled with BioRender.com.