scholarly journals Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide insight into Copper Import

2021 ◽  
Author(s):  
Khalfaoui-Hassani Bahia ◽  
Trasnea Petru-Iulian ◽  
Steimle Stefan ◽  
Koch Hans-Georg ◽  
Fevzi Daldal
Keyword(s):  
Conformational Changes ◽  
Structural Models ◽  
Major Facilitator Superfamily ◽  
Transmembrane Helices ◽  
Surface Loop ◽  
Major Facilitator ◽  
Copper Import ◽  
Cu Uptake ◽  
Insight Into

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS), and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12 transmembrane helices (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role, and remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His and Cys residues of CcoA we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggested a model linking Cu import for cbb3-Cox biogenesis with a thiol: disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function.

The human iron exporter ferroportin. Insight into the transport mechanism by molecular modeling

2016 ◽  
Vol 12 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Valentina Tortosa ◽  
Maria Carmela Bonaccorsi di Patti ◽  
Giovanni Musci ◽  
Fabio Polticelli
Keyword(s):  
Molecular Modeling ◽  
Transport Mechanism ◽  
Structural Models ◽  
Degradation Pathway ◽  
Atomic Level ◽  
Major Facilitator Superfamily ◽  
Wild Type ◽  
Major Facilitator ◽  
First Time ◽  
Insight Into

AbstractFerroportin, a membrane protein belonging to the major facilitator superfamily of transporters, is the only vertebrate iron exporter known so far. Several ferroportin mutations lead to the so-called ferroportin disease or type 4 hemochromatosis, characterized by two distinct iron accumulation phenotypes depending on whether the mutation affects the activity of the protein or its degradation pathway. Through extensive molecular modeling analyses using the structure of all known major facilitator superfamily members as templates, multiple structural models of ferroportin in the three mechanistically relevant conformations (inward open, occluded, and outward open) have been obtained. The best models, selected on the ground of experimental data available on wild-type and mutant ferroportion, provide for the first time a prediction at the atomic level of the dynamics of the transporter. Based on these results, a possible mechanism for iron export is proposed.

scholarly journals Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import

mBio ◽  
2021 ◽  
Author(s):  
Bahia Khalfaoui-Hassani ◽  
Petru-Iulian Trasnea ◽  
Stefan Steimle ◽  
Hans-Georg Koch ◽  
Fevzi Daldal
Keyword(s):  
Oxidative Stress ◽  
Molecular Mechanism ◽  
Major Facilitator Superfamily ◽  
Cellular Homeostasis ◽  
Redox Active ◽  
Major Facilitator ◽  
Copper Import ◽  
Insight Into

Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.

scholarly journals Pyridoxal-5′-phosphate–dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi

2019 ◽  
Vol 117 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Guang Zhi Dai ◽  
Wen Bo Han ◽  
Ya Ning Mei ◽  
Kuang Xu ◽  
Rui Hua Jiao ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Complex Structure ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Major Facilitator Superfamily ◽  
Molecular Architecture ◽  
Widespread Occurrence ◽  
Indolizidine Alkaloids ◽  
Major Facilitator ◽  
Insight Into

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C–C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A−I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature’s arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.

scholarly journals Two Nitrate/Nitrite Transporters Are Encoded within the Mobilizable Plasmid for Nitrate Respiration of Thermus thermophilus HB8

2000 ◽  
Vol 182 (8) ◽  
pp. 2179-2183 ◽  
Author(s):  
Sandra Ramírez ◽  
Renata Moreno ◽  
Olga Zafra ◽  
Pablo Castán ◽  
Cristina Vallés ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Average Distance ◽  
Thermus Thermophilus ◽  
Major Facilitator Superfamily ◽  
Nitrate Respiration ◽  
Mobilizable Plasmid ◽  
Excretion Rates ◽  
Major Facilitator ◽  
Nitrate And Nitrite

ABSTRACT Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.

scholarly journals Structure of Macrolide Efflux Protein (MefA/E) in Streptococcus pneumoniae: An in silico approach

2020 ◽  
Author(s):  
Sreeram Chandra Murthy Peela ◽  
Jyoti Sharma ◽  
Sujatha Sistla
Keyword(s):  
Streptococcus Pneumoniae ◽  
Respiratory Tract ◽  
Protein Sequence ◽  
In Silico ◽  
Respiratory Tract Infections ◽  
Major Facilitator Superfamily ◽  
Transmembrane Helices ◽  
E Protein ◽  
Major Facilitator ◽  
Tract Infections

AbstractBackgroundMacrolides are one of the commonest antibiotics used to treat bacterial respiratory tract infections. Resistance to this class of antibiotics is on the rise and is mediated by macrolide efflux (MefA/E) protein as one of the mechanisms. Despite its importance, the structure of this protein is not known yet.MethodsThe publicly available MefA/E protein sequence was used to model the structure. Modelling was performed in I-TASSER, and the model was further refined. Its orientation in a membrane was studied using OPM server.Results and conclusionsThe structure of MefA/E resembled that of Major Facilitator Superfamily (MFS) proteins, with 13 transmembrane helices. It had a V-shaped conformation, with the wider part towards the outer membrane layer.

scholarly journals It takes two to tango: The dance of the permease

2019 ◽  
Vol 151 (7) ◽  
pp. 878-886 ◽  
Author(s):  
H. Ronald Kaback ◽  
Lan Guan
Keyword(s):  
Binding Sites ◽  
Cytoplasmic Membrane ◽  
Major Facilitator Superfamily ◽  
Internal Cavity ◽  
Lactose Permease ◽  
Physiological Conditions ◽  
Membrane Transport Proteins ◽  
Major Facilitator ◽  
Alternating Access ◽  
Insight Into

The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H+ are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H+ symport in either direction across the cytoplasmic membrane. The H+-binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient.

Active transporters as enzymes: an energetic framework applied to major facilitator superfamily and ABC importer systems

2015 ◽  
Vol 467 (2) ◽  
pp. 193-199 ◽  
Author(s):  
Brian H. Shilton
Keyword(s):  
Active Transport ◽  
Conformational Changes ◽  
Transport Process ◽  
Energy Use ◽  
High Energy ◽  
Major Facilitator Superfamily ◽  
Transport Systems ◽  
Solute Movement ◽  
Major Facilitator ◽  
Membrane Transport Systems

Active membrane transporters are dynamic molecular machines that catalyse transport across a membrane by coupling solute movement to a source of energy such as ATP or a secondary ion gradient. A central question for many active transporters concerns the mechanism by which transport is coupled to a source of energy. The transport process and associated energetic coupling involve conformational changes in the transporter. For efficient transport, the conformational changes must be tightly regulated and they must link energy use to movement of the substrate across the membrane. The present review discusses active transport using the well-established energetic framework for enzyme-mediated catalysis. In particular, membrane transport systems can be viewed as ensembles consisting of low-energy and high-energy conformations. The transport process involves binding interactions that selectively stabilize the higher energy conformations, and in this way promote conformational changes in the system that are coupled to decreases in free energy and substrate translocation. The major facilitator superfamily of secondary active transporters is used to illustrate these ideas, which are then be expanded to primary active transport mediated by ABC (ATP-binding cassette) import systems, with a focus on the well-studied maltose transporter.

scholarly journals Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane

2008 ◽  
Vol 190 (7) ◽  
pp. 2441-2449 ◽  
Author(s):  
Karl A. Hassan ◽  
Talal Souhani ◽  
Ronald A. Skurray ◽  
Melissa H. Brown
Keyword(s):  
Major Facilitator Superfamily ◽  
Tryptophan Residue ◽  
Transmembrane Helices ◽  
Multidrug Efflux ◽  
Long Distance ◽  
Structural Requirement ◽  
Tryptophan Residues ◽  
Functional Consequences ◽  
Major Facilitator ◽  
Cytoplasmic Loops

ABSTRACT Tryptophan residues can possess a multitude of functions within a multidrug transport protein, e.g., mediating interactions with substrates or distal parts of the protein, or fulfilling a structural requirement, such as guiding the depth of membrane insertion. In this study, the nine tryptophan residues of the staphylococcal QacA multidrug efflux protein were individually mutated to alanine and phenylalanine, and the functional consequences of these changes were determined. Phenylalanine substitutions for each tryptophan residue were functionally tolerated. However, alanine modifications revealed an important functional role for three tryptophan residues, W58, W149, and W173, each of which is well conserved among QacA-related transport proteins in the major facilitator superfamily. The most functionally compromising mutation, an alanine substitution for W58, likely to be located at the extracellular interface of transmembrane segment 2, abolished all detectable QacA-mediated resistance and transport function. Second-site suppressor analyses identified several mutations that rescued the function of the W58A QacA mutant. Remarkably, all of these suppressor mutations were shown to be located in cytoplasmic loops between transmembrane helices 2 and 3 or 12 and 13, demonstrating novel functional associations between amino acid positions on opposite sides of the membrane and in distal N- and C-terminal regions of the QacA protein.

scholarly journals Conformational Transition Pathways in Major Facilitator Superfamily Transporters

2019 ◽  
Author(s):  
Dylan Ogden ◽  
Kalyan Immadisetty ◽  
Mahmoud Moradi
Keyword(s):  
Conformational Changes ◽  
Glucose Transporter ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Salt Bridge ◽  
Md Simulations ◽  
Major Facilitator Superfamily ◽  
Glucose Transporter 1 ◽  
Major Facilitator ◽  
Mfs Transporters

AbstractMajor facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of MFS superfamily. We have performed a variety of equilibrium, non-equilibrium, biased, and unbiased all-atom molecular dynamics (MD) simulations of bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.

scholarly journals Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

2021 ◽  
Author(s):  
Elisabeth Lambert ◽  
Ahmad Reza Mehdipour ◽  
Alex Schmidt ◽  
Gerhard Hummer ◽  
Camilo Perez
Keyword(s):  
Lipid Binding ◽  
Major Facilitator Superfamily ◽  
Common Mechanism ◽  
Lipid Transporters ◽  
Dynamics Simulations ◽  
Major Facilitator ◽  
Lipid Transporter ◽  
Lipid Translocation

Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a trap-and-flip mechanism that might be shared among MFS lipid transporters.

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