The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli

King, S. Bruce; Hansen, Camilla Hartmann Friis; Wilson, Thomas H.

doi:10.1016/0005-2736(91)90390-t

Cited by 136 publications

(126 citation statements)

References 27 publications

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“…Second-site revertants of D237N had a mutation that converted Lys-358 to Gln-358, whereas revertants of K358T contained a neutral amino acid (Asn, Gly or Tyr) at position 237. The second-site revertants exhibit galactoside accumulation and have activities that are 30-60% the rate of the wild-type [143]. From these studies it is concluded that Asp-237 (helix VII) and Lys-358 (helix XI) are closely juxtaposed in the three-dimensional structure, and possibly form a charge-neutralizing salt bridge.…”

Section: Iv-b Tertiary Structurementioning

confidence: 93%

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Secondary solute transport in bacteria

Poolman

Konings

1993

Biochimica et Biophysica Acta (BBA) - Bioenergetics

199

160

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Section: Iv-b Tertiary Structurementioning

confidence: 93%

“…King et al [143] were the first to isolate second-site revertants of Asp-237 (D237) and Lys-358 (K358) in the lactose carrier of E. coli. Mutagenesis of each of these residues results in a defect in transport.…”

Section: Iv-b Tertiary Structurementioning

confidence: 99%

Secondary solute transport in bacteria

Poolman

Konings

1993

Biochimica et Biophysica Acta (BBA) - Bioenergetics

199

160

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“…However, Ala122 does not make direct contact with TDG either. Asp240 (helix VII) and Lys319 (helix X) interact relatively weakly, and mutants with double-neutral replacements (Cys or Ala) exhibit low but significant ability to catalyze lactose accumulation (29)(30)(31).…”

Section: Structural Evidence For An Occluded Intermediatementioning

confidence: 99%

A chemiosmotic mechanism of symport

Kaback

2015

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

119

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Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H + across the Escherichia coli membrane (galactoside/H + symport). Initial X-ray structures reveal N-and C-terminal domains, each with six largely irregular transmembrane helices surrounding an aqueous cavity open to the cytoplasm. Recently, a structure with a narrow periplasmic opening and an occluded galactoside was obtained, confirming many observations and indicating that sugar binding involves induced fit. LacY catalyzes symport by an alternating access mechanism. Experimental findings garnered over 45 y indicate the following: (i) The limiting step for lactose/H + symport in the absence of the H + electrochemical gradient (Δμ̃H+) is deprotonation, whereas in the presence of Δμ̃H+, the limiting step is opening of apo LacY on the other side of the membrane; (ii) LacY must be protonated to bind galactoside (the pK for binding is ∼10.5); (iii) galactoside binding and dissociation, not Δμ̃H+, are the driving forces for alternating access; (iv) galactoside binding involves induced fit, causing transition to an occluded intermediate that undergoes alternating access; (v) galactoside dissociates, releasing the energy of binding; and (vi) Arg302 comes into proximity with protonated Glu325, causing deprotonation. Accumulation of galactoside against a concentration gradient does not involve a change in K d for sugar on either side of the membrane, but the pK a (the affinity for H + ) decreases markedly. Thus, transport is driven chemiosmotically but, contrary to expectation, ΔμH+ acts kinetically to control the rate of the process.The lactose permease of Escherichia coli (LacY) specifically binds and catalyzes symport of D-Gal and D-galactopyranosides with an H + (galactoside/H + symport), but it does not recognize the analogous glucopyranosides, which differ only in the orientation of the C4-OH of the pyranosyl ring (reviewed in refs. 1, 2). Typical of many major facilitator superfamily (MFS) members, LacY couples the free energy released from downhill translocation of H + in response to an H + electrochemical gradient (ΔμH+) to drive accumulation of galactopyranosides against a concentration gradient. Because coupling between sugar and H + translocation is obligatory, in the absence of Δμ̃H+, LacY can also transduce the energy released from the downhill transport of sugar to drive uphill H + transport with the generation of Δμ̃H+, the polarity of which depends upon the direction of the sugar gradient. However, the mechanism by which this so-called "chemiosmotic" process occurs remains obscure. This contribution aims at clarifying the specific steps underpinning the mechanism of galactoside/ H + symport.

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“…Arginine and lysine residues through electrostatic interactions with anionic residues are important for the structure of membrane-spanning domains in other proteins such as the Lac permease and the inward-rectified K ϩ channel, IRK1 (33)(34)(35). As discussed by Perutz (18), salt bridges within proteins are an important structural feature that confers thermostability and resistance to denaturation.…”

Section: Voltage Dependence Of O L and O B -Tomentioning

confidence: 99%

Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

Cotten

Welsh

1999

Journal of Biological Chemistry

107

105

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Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wildtype CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction. The cystic fibrosis transmembrane conductance regulator (CFTR)1 contains an anion-selective pore (1-6). Earlier work has shown that amino acid residues in the two membranespanning domains, MSD1 and MSD2, determine the properties of this pore and harbor a number of disease-associated mutations (7-12). Despite the importance of this region to CFTR function, an understanding of the pore and MSD structure is limited.Studies of the effect of cystic fibrosis (CF)-associated mutations have been of value in identifying structurally and functionally important regions of CFTR. At least four CF-associated mutations have been identified at position 347 in M6: R347C, R347H, R347L, and R347P, suggesting that Arg-347 is important for CFTR structure and function (13-15).2 Early studies by Sheppard et al. (7) showed that mutation of Arg-347 to proline significantly decreased single-channel conductance with little effect on CFTR trafficking to the plasma membrane. Other work by Tabcharani et al. (8,16) and Linsdell and Hanrahan (17) emphasized the importance of Arg-347 for anomalous mole-fraction behavior, iodide permeability, and voltagedependent block by DIDS, in addition to single-channel conductance. Interestingly, mutation of Arg-347 to a histidine (R347H) produced a channel that displayed pH-dependent conductance and anomalous mole-fraction behavior (8). These studies suggested that Arg-347 may line the pore and that a positive charge at position 347 is sufficient for wild-type conductance. Because mutation of Arg-347 eliminated anomalous mole-fraction behavior, Arg-347 itself was proposed to be an anion binding site in the CFTR pore (8), and the presumed positive charge introduced upon protonation of His-347 was thought to facilitate interactions with permeating a...

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The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli

Cited by 136 publications

References 27 publications

Secondary solute transport in bacteria

Secondary solute transport in bacteria

A chemiosmotic mechanism of symport

Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

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