Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Markham, Kelsey J.; Tikhonova, Elena B.; Scarpa, Aaron C.; Hariharan, Parameswaran; Katsube, Satoshi; Guan, Lan

doi:10.1016/j.jbc.2021.101090

Cited by 13 publications

(12 citation statements)

References 52 publications

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“…The results of melibiose active transport mediated by the WT MelB St and the mutants D55C and D59C have been published previously (7,8,19). The data are reproducible and re-plotted for comparison with the melibiose fermentation data.…”

Section: Melibiose Transport Activitymentioning

confidence: 82%

“…There are two well-conserved acidic residues in the cation-binding pocket of MelB, and those two positions have also been well-studied in the past by several laboratories using varied methods (5,7,8,17,19,20). Cys or Ala mutants on the strictly conserved Asp59 lose the binding of Na + and Li + , as well as the melibiose active transport coupled to the Na + , Li + , or H + .…”

Section: Discussionmentioning

confidence: 99%

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Distinct roles of the major binding residues in the cation-binding pocket of MelB

Hariharan,

Bakhtiiari,

Liang

et al. 2024

Preprint

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Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with either H+, Li+, or Na+, but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt, as well as the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelBSt cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published previously. We determined the energetic contributions of three major Na+-binding residues in cation selectivity for Na+ and H+ by the free energy simulations. The D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport with poor activities at higher delta pH and better activities at reversal delta pH was observed, supporting that the membrane potential is the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket.

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Section: Melibiose Transport Activitymentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Distinct roles of the major binding residues in the cation-binding pocket of MelB

Hariharan,

Bakhtiiari,

Liang

et al. 2024

Preprint

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“…The core of the inner barrier is formed by three-helix pairs from both domains (V/VIII, IV/X, and back II/XI), and stabilized by an extensive charged network ( Fig. 4b ) 11,16,19,48 . The N-terminal Arg141 (helix-V) forms salt-bridge interactions with the C-terminal Asp351 and Asp354 (helix-X), and this core interaction links with four other charged residues to stabilize the cytoplasmic closure by forming an inner barrier.…”

Section: Resultsmentioning

confidence: 99%

“…Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is the prototype Na + /solute transporter of the major facilitator superfamilies (MFS) 11,[15][16][17][18][19][20][21][22][23][24] . It catalyzes the stoichiometric symport of galactopyranoside with Na + , H + , or Li +25 .…”

Section: Introductionmentioning

confidence: 99%

Mobile barrier mechanisms for Na⁺-coupled symport in an MFS sugar transporter

Hariharan,

Shi,

Katsube

et al. 2023

Preprint

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While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined an inward-facing Na+-bound, low-sugar affinity cryoEM structure. It is the first structure of a major facilitator superfamily (MFS) transporter with experimentally determined cation binding, and also a structure mimicking the physiological regulatory state of MelB under the global regulator EIIAGlc of the glucose-specific phosphoenolpyruvate:phosphotransferase system. Collectively with the available outward-facing sugar-bound structures and a large body of functional analysis, we identified that only inner barrier that exists in the outward-facing conformation contributes to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-facing conformation, the sugar selectivity pocket is also broken, resulting in a decreased sugar-binding affinity by greater than 30-fold, which can facilitate the substrate release and accumulation intracellularly. The inner/outer barrier shifting directly regulates the sugar-binding affinity, with no effect on the cation binding as also suggested by molecular dynamics simulations. Furthermore, the use of the inward-facing conformation-specific Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions linked to the inner barrier-specific charged network and critical for the barrier switching mechanisms. The complementary results provided structural, dynamic, and regulatory insights into the mobile barrier mechanism for cation-coupled symport.

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“…5H ), also previously used, is less sensitive than Ala scanning, but has the advantage that Cys can be crosslinked or readily covalently modified. [ 59 , 60 ]…”

Section: Discussionmentioning

confidence: 99%

GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform

et al. 2022

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Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Cited by 13 publications

References 52 publications

Distinct roles of the major binding residues in the cation-binding pocket of MelB

Distinct roles of the major binding residues in the cation-binding pocket of MelB

Mobile barrier mechanisms for Na⁺-coupled symport in an MFS sugar transporter

GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform

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Complete cysteine-scanning mutagenesis of the Salmonella typhimurium melibiose permease

Cited by 13 publications

References 52 publications

Distinct roles of the major binding residues in the cation-binding pocket of MelB

Distinct roles of the major binding residues in the cation-binding pocket of MelB

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter

GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform

Contact Info

Product

Resources

About

Mobile barrier mechanisms for Na⁺-coupled symport in an MFS sugar transporter