By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. ...A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family's extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.
The small multidrug resistance (SMR) family of membrane proteins is prominent because of its rare dual topology architecture, simplicity, and small size. Its best studied member, EmrE, is an ...important model system in several fields related to membrane protein biology, from evolution to mechanism. But despite decades of work on these multidrug transporters, the native function of the SMR family has remained a mystery, and many highly similar SMR homologs do not transport drugs at all. Here we establish that representative SMR proteins, selected from each of the major clades in the phylogeny, function as guanidinium ion exporters. Drug-exporting SMRs are all clustered in a single minority clade. Using membrane transport experiments, we show that these guanidinium exporters, which we term Gdx, are very selective for guanidinium and strictly and stoichiometrically couple its export with the import of two protons. These findings draw important mechanistic distinctions with the notably promiscuous and weakly coupled drug exporters like EmrE.
Insertions and deletions (indels) enable evolution and cause disease. Due to technical challenges, indels are left out of most mutational scans, limiting our understanding of them in disease, ...biology, and evolution. We develop a low cost and bias method, DIMPLE, for systematically generating deletions, insertions, and missense mutations in genes, which we test on a range of targets, including Kir2.1. We use DIMPLE to study how indels impact potassium channel structure, disease, and evolution. We find deletions are most disruptive overall, beta sheets are most sensitive to indels, and flexible loops are sensitive to deletions yet tolerate insertions.
The small multidrug resistance (SMR) family is composed of widespread microbial membrane proteins that fulfill different transport functions. Four functional SMR subtypes have been identified, which ...variously transport the small, charged metabolite guanidinium, bulky hydrophobic drugs and antiseptics, polyamines, and glycolipids across the membrane bilayer. The transporters possess a minimalist architecture, with ∼100-residue subunits that require assembly into homodimers or heterodimers for transport. In part because of their simple construction, the SMRs are a tractable system for biochemical and biophysical analysis. Studies of SMR transporters over the last 25 years have yielded deep insights for diverse fields, including membrane protein topology and evolution, mechanisms of membrane transport, and bacterial multidrug resistance. Here, we review recent advances in understanding the structures and functions of SMR transporters. New molecular structures of SMRs representing two of the four functional subtypes reveal the conserved structural features that have permitted the emergence of disparate substrate transport functions in the SMR family and illuminate structural similarities with a distantly related membrane transporter family, SLC35/DMT.
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•Flucs are highly selective fluoride channels that protect microbes against cytoplasmic accumulation of toxic fluoride ion.•Flucs are found as dual-topology homodimers, heterodimers, ...and fused 2-domain proteins with inverted topologies.•Crystal structures of dual-topology Flucs show a symmetrical protein with two pores and a buried Na+ ion at the dimer interface.•Both pores function in the homodimers, but in heterodimers and 2-domain fused proteins, the second pore has degraded.
Dual-topology proteins are likely evolutionary antecedents to a common motif in membrane protein structures, the inverted repeat. A family of fluoride channels, the Flucs, which protect microorganisms, fungi, and plants against cytoplasmic fluoride accumulation, has representatives of all topologies along this evolutionary trajectory, including dual-topology homodimers, antiparallel heterodimers, and, in eukaryotes, fused two-domain proteins with an inverted repeat motif. Recent high-resolution crystal structures of dual-topology homodimers, coupled with extensive functional information about both the homodimers and two-domain Flucs, provide a case study of the co-evolution of fold and function.
Mutations in transporters can impact an individual’s response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine ...saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.
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•Deep mutational scan of OCT1 reveals the determinants of biogenesis and substrate uptake•Discovery of a conserved motif, the stability helix, essential to SLC22 biogenesis•AI structure prediction and mutational data for accurate structure-function modeling•Integrating genomic-health records reveals variants effect on human physiology
Yee et al. create a complete functional map of how genetic variants in the OCT1 transporter alter biogenesis and substrate uptake. This work provides a framework for biophysically informed precision medicine by integrating functional genomics, mechanistic modeling, and human genetics to predict variant effects on disease and drug efficacy.
Enzyme‐mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and ...straightforward strategy for the efficient capture and recycling of enzymes using a small‐molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N‐terminally labeled with lithocholic acid (LA)—an inexpensive bile acid that exhibits strong binding to β‐cyclodextrin (βCD). Capture and recycling of the LA‐Pro‐SrtA 7M conjugate was achieved using βCD‐modified sepharose resin. The LA‐Pro‐SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.
Reuse, recycle: A scalable and straightforward strategy is demonstrated for the efficient capture and recycling of enzymes using a small‐molecule affinity tag. A proline variant of an evolved sortase A was labeled with lithocholic acid (LA). Capture and recycling of this bioconjugate from reaction mixtures was achieved using a βCD‐modified sepharose resin (βCD=β‐cyclodextrin).
The first 23-step total synthesis of the cyclodepsipeptide dolastatin 16 (1) has been achieved. Synthesis of the dolaphenvaline and dolamethylleuine amino acid units using simplified methods improved ...the overall efficiency. The formation of the 25-membered macrocycle employing lactonization with 2-methyl-6-nitrobenzoic anhydride completed a key step in the synthesis. Regrettably, the synthetic dolastatin 16 (1), while otherwise identical (by X-ray crystal structure and spectral analyses) with the natural product, did not reproduce the powerful (nanomolar) cancer cell growth inhibition displayed by the natural isolate. Presumably this result can be attributed to conformation(s) of the synthetic dolastatin 16 (1) or to a chemically undetected component isolated with the natural product.
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