Na+/Melibiose Membrane Transport Protein, MelB

Guan, Lan

doi:10.1007/978-3-642-35943-9_10082-1

Cited by 12 publications

(12 citation statements)

References 11 publications

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“…It is well expressed in E. coli and purified protein is stable. MelBSt and the E. coli MelB (MelBEc) have been subjected to varied biochemical and biophysical studies including x-ray crystallographic analyses, as well as the development of novel analytical techniques for membrane protein research (6,7,(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The 3D crystal structures of MelBSt confirmed the previous computational predictions (19,24).…”

Section: Introductionsupporting

confidence: 64%

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Markham

Tikhonova

Scarpa

et al. 2021

Journal of Biological Chemistry

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Section: Introductionsupporting

confidence: 64%

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Markham

Tikhonova

Scarpa

et al. 2021

Journal of Biological Chemistry

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“…The functions of cell membranes rely on both lipids and proteins, as well as their interactions. Salmonella typhimurium MelB (MelB St ) encoded by the melAB operon is a cation-coupled symporter with 12 transmembrane α-helices embedded in the cytoplasmic membrane [ 1 – 3 ]. This transporter catalyzes stoichiometric melibiose translocation across the membrane coupled to the transduction of the cations Na + , Li + , or H + [ 1 , 2 , 4 – 6 ].…”

Section: Introductionmentioning

confidence: 99%

“…Salmonella typhimurium MelB (MelB St ) encoded by the melAB operon is a cation-coupled symporter with 12 transmembrane α-helices embedded in the cytoplasmic membrane [ 1 – 3 ]. This transporter catalyzes stoichiometric melibiose translocation across the membrane coupled to the transduction of the cations Na + , Li + , or H + [ 1 , 2 , 4 – 6 ]. Melibiose active transport against concentration gradient is driven by an electrochemical H + , Na + , or Li + gradient, while MelB can also catalyze melibiose downhill transport in the absence of these electrochemical ion gradients.…”

Section: Introductionmentioning

confidence: 99%

“…1 ), and no lipid was resolved. An alternating-access mechanism was suggested to operate the transport by MelB [ 1 , 2 , 11 – 14 ], i.e., the permeases cycle different conformational states including at least an outward-open, inward-open, and occluded intermediate state. A recent study suggests that the cooperative binding of the cargo sugar and the coupling cation to MelB St triggers the global conformational changes [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Structural and functional characterization of protein–lipid interactions of the Salmonella typhimurium melibiose transporter MelB

et al. 2018

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BackgroundMembrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein.ResultsLipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2–3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy.Conclusions(1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0553-0) contains supplementary material, which is available to authorized users.

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“…The bacterial melibiose permease MelB is one of the few membrane transporters discovered at earlier years (8) , and it has been serving as a model system for the study of cation-coupled transport mechanisms (9)(10)(11)(12) , as well as the development of novel analytical techniques for membrane protein research (11,(13)(14)(15)(16)(17) . It is a well-documented representative from MFS_2 family (1,7,10,12,(18)(19)(20)(21)(22) . MelB catalyzes the stoichiometric galactose or galactosides symport with monovalent cation either Na + , Li + , or H + , with no affinity for glucose or glucosides (23)(24)(25)(26) .…”

Section: /Mainmentioning

confidence: 99%

Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

Guan

Hariharan

2020

Preprint

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The symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, which were refined to a resolution of 3.05 or 3.15 Å, respectively, are virtually identical at an outward-facing conformation; each one contains a α-galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters.SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

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Na+/Melibiose Membrane Transport Protein, MelB

Cited by 12 publications

References 11 publications

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Structural and functional characterization of protein–lipid interactions of the Salmonella typhimurium melibiose transporter MelB

Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

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