Ins and Outs of Major Facilitator Superfamily Antiporters

Law, Christopher J.; Maloney, Peter C.; Wang, Da-Neng

doi:10.1146/annurev.micro.61.080706.093329

Cited by 443 publications

(481 citation statements)

References 85 publications

(136 reference statements)

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“…A similar model has been proposed for a number of other transporters (e.g., refs. [20][21][22][23][24]. It is also likely that the alternating access model for LacY involves formation of an occluded intermediate (25,26), which is consistent with the highly dynamic nature of the protein (15,(27)(28)(29)(30)(31).…”

mentioning

confidence: 67%

Role of the irreplaceable residues in the LacY alternating access mechanism

Zhou¹,

Jiang²,

Kaback

2012

Proc. Natl. Acad. Sci. U.S.A.

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Few side chains in the galactoside/H + symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H + translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H + symport is abolished as well.membrane protein | membranes | protein dynamics | transport T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H + (i.e., galactoside/H + symport). Thus, LacY transduces free energy stored in an H + electrochemical gradient (Δμ H +) into a galactoside concentration gradient. As transport is obligatorily coupled, LacY also transduces free energy stored in a sugar concentration gradient into Δμ H +, the polarity of which depends on the direction of the sugar gradient (reviewed in ref. 1). LacY has been solubilized from the membrane, purified to homogeneity in a highly functional state (2), and is structurally (3, 4), as well as functionally (5), a monomer.X-ray crystal structures of LacY (6-9) and various independent biochemical and spectroscopic findings (10-17) provide converging evidence for an alternating access mechanism. By this means, sugar binding induces coordinated opening and closing of periplasmic and cytoplasmic cavities, respectively, thereby allowing accessibility of sugar-and H + -binding sites to either side of the membrane (i.e., the alternating access model; reviewed in refs. 18, 19). A similar model has been proposed for a number of other transporters (e.g., refs. 20-24). It is also likely that the alternating access model for LacY involves formation of an occluded intermediate (25,26), which is consistent with the highly dynamic nature of the protein (15, 27-31).Cys-scanning and site-directed mutagenesis of each residue in LacY de...

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mentioning

confidence: 67%

Role of the irreplaceable residues in the LacY alternating access mechanism

Zhou¹,

Jiang²,

Kaback

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Studies with other MFS transporters, however, indicate that the interdomain loop is necessary for movement and flexibility between the N‐ and C‐terminal domains to facilitate the pumping mechanism required for substrate translocation (Law et al , 2008; Dang et al , 2010). …”

Section: Resultsmentioning

confidence: 99%

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

Perez

Gomez

Kalvapalle

et al. 2017

Molecular Systems Biology

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The major facilitator superfamily (MFS) effluxers are prominent mediators of antimicrobial resistance. The biochemical characterization of MFS proteins is hindered by their complex membrane environment that makes in vitro biochemical analysis challenging. Since the physicochemical properties of proteins drive the fitness of an organism, we posed the question of whether we could reverse that relationship and derive meaningful biochemical parameters for a single protein simply from fitness changes it confers under varying strengths of selection. Here, we present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the MFS tetracycline efflux pump, TetB, and a family of single amino acid variants. We determined two lumped biochemical parameters roughly describing K m and V max for TetB and variants. Including in vivo protein levels into our model allowed for more specified prediction of pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants. We further demonstrated the general utility of our model by solely using fitness to assay a library of tet(B) variants and estimate their biochemical properties.

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“…Another carbohydrate-related function highly enriched in the honey bee microbiome and detected in all bins, was the family of "arabinose efflux permease." Proteins of this COG function belong to the major facilitator superfamily responsible for the import or export of a broad range of different substrates, including sugars, drugs, and peptides (29). We found a high diversity among these proteins in our metagenomic data with a marked number showing homology to drug resistance efflux pumps (SI Appendix, Fig.…”

mentioning

confidence: 87%

Functional diversity within the simple gut microbiota of the honey bee

Engel

Martinson

Moran

2012

Proc. Natl. Acad. Sci. U.S.A.

723

775

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Animals living in social communities typically harbor a characteristic gut microbiota important for nutrition and pathogen defense. Accordingly, in the gut of the honey bee, Apis mellifera , a distinctive microbial community, composed of a taxonomically restricted set of species specific to social bees, has been identified. Despite the ecological and economical importance of honey bees and the increasing concern about population declines, the role of their gut symbionts for colony health and nutrition is unknown. Here, we sequenced the metagenome of the gut microbiota of honey bees. Unexpectedly, we found a remarkable degree of genetic diversity within the few bacterial species colonizing the bee gut. Comparative analysis of gene contents suggests that different species harbor distinct functional capabilities linked to host interaction, biofilm formation, and carbohydrate breakdown. Whereas the former two functions could be critical for pathogen defense and immunity, the latter one might assist nutrient utilization. In a γ-proteobacterial species, we identified genes encoding pectin-degrading enzymes likely involved in the breakdown of pollen walls. Experimental investigation showed that this activity is restricted to a subset of strains of this species providing evidence for niche specialization. Long-standing association of these gut symbionts with their hosts, favored by the eusocial lifestyle of honey bees, might have promoted the genetic and functional diversification of these bee-specific bacteria. Besides revealing insights into mutualistic functions governed by the microbiota of this important pollinator, our findings indicate that the honey bee can serve as a model for understanding more complex gut-associated microbial communities.

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Ins and Outs of Major Facilitator Superfamily Antiporters

Cited by 443 publications

References 85 publications

Role of the irreplaceable residues in the LacY alternating access mechanism

Role of the irreplaceable residues in the LacY alternating access mechanism

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

Functional diversity within the simple gut microbiota of the honey bee

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