Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

Zani, M L; Pourcher, Thierry; Leblanc, Gérard

doi:10.1016/s0021-9258(17)31473-4

Cited by 48 publications

(33 citation statements)

References 35 publications

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“…5a, b , Supplementary Fig. 7a ), which confirms the previous conclusion based on a large body of biochemical/biophysical analyses including mutagenesis and second-site suppressors, all favoring the overlapping sites for sugars and cations 36 , 50 , 51 , 53 , 61 , 62 . While the Na + binding was not resolved with MelB, as mentioned above, several positions including 55, 59, 117 and 121 have been determined essential or important for the binding of Na + and Li + , as well as for the coupled transport 9 , 24 , 36 – 38 , 46 , 47 , 63 , 64 .…”

Section: Discussionsupporting

confidence: 89%

“…Mutations on most of these positions largely reduce the binding or transport in MelB St and MelB Ec , and some mutants also affected affinity to both sugar and cation 9 , 36 , 50 – 53 .…”

Section: Resultsmentioning

confidence: 99%

“…The helices II (Lys18, Asp19, Ile22, and Tyr26, particularly Asp19 and Lys18) and VI (Tyr120, Asp124, and Trp128, particularly Asp124) make major contributions to the sugar binding, and helices V (Arg149), X (Trp342), and XI (Thr373 and Val376) also contribute to the binding affinity. Mutations on most of these positions greatly reduce the binding or transport in MelB, and some affected affinity to both sugar and cation 9 , 36 , 50 – 53 . The structure was also strongly supported by the Fourier-transform infrared spectroscopy (FTIR) studies in MelB Ec , where the mutants on position-19 or 124 lost sugar-specific FTIR signals, and the D124C mutant also showed greatly reduced Na + -specific signals 36 .…”

Section: Discussionmentioning

confidence: 99%

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X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Guan

Hariharan

2021

Commun Biol

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Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

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Section: Discussionsupporting

confidence: 89%

“…Mutations on most of these positions largely reduce the binding or transport in MelB St and MelB Ec , and some mutants also affected affinity to both sugar and cation 9 , 36 , 50 – 53 .…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Guan

Hariharan

2021

Commun Biol

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“…1) and the sugar-binding site is delimited at least in part by C-terminal domains (darker gray helices in Fig. 1); 2), helix IV may connect the two substrate-binding sites (18), and loops 4-5 and 10-11 are important for MelB function (8,(19)(20)(21); and 3), Asp and Glu residues are implicated in cation and melibiose binding and/or translocation (16,20,(22)(23)(24). Other residues, such as Asn (25), Arg (19,26), and Tyr (24), have also been shown to be important in these processes.…”

Section: Introductionmentioning

confidence: 99%

Alteration of Sugar-Induced Conformational Changes of the Melibiose Permease by Mutating Arg141 in Loop 4-5

León¹,

Leblanc²,

Padrós³

2009

Biophysical Journal

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The melibiose permease (MelB) from Escherichia coli couples the uptake of melibiose to that of Na+, Li+, or H+. In this work, we applied attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy to obtain information about the structural changes involved in substrate interaction with the R141C mutant and with the wild-type MelB reacted with N-ethylmaleimide (NEM). These modified permeases have the ability to bind the substrates but fail to transport them. It is shown that the sugar-induced ATR-FTIR difference spectra of the R141C mutant are different from those corresponding to the Cys-less permease from which it is derived. There are alterations of peaks assigned to turns and beta-structures located most likely in loop 4-5. In addition, and quite notably, a peak at 1659 cm(-1), assigned to changes at the level of one alpha-helix subpopulation, disappears in the melibiose-induced difference spectrum in the presence of Na+, suggesting a reduction of the conformational change capacity of the mutated MelB. These helices may involve structural components that couple the cation- and sugar-binding sites. On the other hand, MelB-NEM difference spectra are proportionally less disrupted than the R141C ones. Hence, the transport cycle of these two permeases, modified at two different loops, is most likely impaired at a different stage. It is proposed that the R141C mutant leads to the generation of a partially defective ternary complex that is unable to catalyze the subsequent conformational change necessary for substrate translocation.

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“…It would be interesting to test if the D35E could also rescue melibiose transport of D351C, D354C, or other inactive mutants in this important area. In MelBEc, these Asp residues have been also showed to be important for melibiose transport activities (26,41,43,48).…”

Section: Discussionmentioning

confidence: 99%

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Markham

Tikhonova

Scarpa

et al. 2021

Journal of Biological Chemistry

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show abstract

Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

Cited by 48 publications

References 35 publications

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

Alteration of Sugar-Induced Conformational Changes of the Melibiose Permease by Mutating Arg141 in Loop 4-5

Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

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