Biomedical subjects

Lan Guan

Publications and source records attributed to Lan Guan.

14 recordsLinked to original sources

PubMed2006

Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY.

Cation-coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of Escherichia coli determined at acidic and neutral pH, and propose a model for the mechanism of coupling between lactose and H+ translocation. No sugar-binding site is observed in the absence of ligand, and deprotonation of the key residue Glu269 is associated with ligand binding. Thus, substrate induces formation of the sugar-binding site, as well as the initial step in H+ transduction.

Binding Sites↗

PubMed2006

Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.

High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knock-out E. coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P.

Aminopeptidases↗

PubMed2006

Manipulating phospholipids for crystallization of a membrane transport protein.

Crystallization is a major bottleneck to obtaining x-ray structures of membrane proteins. By applying an established crystallization protocol for the lactose permease (LacY) of Escherichia coli, a systematic study of the effect of phospholipids (PL) on crystallization of LacY was undertaken. We observe three different crystal forms that diffract to increasingly better resolution in a manner that correlates with the concentration of copurified PL. Consistently, progressive addition of E. coli PL to delipidated LacY leads to different crystal forms. Tetragonal crystals are obtained with improved diffraction quality for a stable mutant by carefully adjusting PL content. Furthermore, crystals of good quality from wild-type LacY, a particularly difficult protein, were also obtained by using same approach. Thus, it is likely that manipulation of PL is a good strategy for predominantly hydrophobic membrane proteins like LacY.

Chromatography, Gel↗

PubMed2006

Lessons from lactose permease.

An X-ray structure of the lactose permease of Escherichia coli (LacY) in an inward-facing conformation has been solved. LacY contains N- and C-terminal domains, each with six transmembrane helices, positioned pseudosymmetrically. Ligand is bound at the apex of a hydrophilic cavity in the approximate middle of the molecule. Residues involved in substrate binding and H+ translocation are aligned parallel to the membrane at the same level and may be exposed to a water-filled cavity in both the inward- and outward-facing conformations, thereby allowing both sugar and H+ release directly into either cavity. These structural features may explain why LacY catalyzes galactoside/H+ symport in both directions utilizing the same residues. A working model for the mechanism is presented that involves alternating access of both the sugar- and H+-binding sites to either side of the membrane.

Binding Sites↗

PubMed2004

Sugar recognition by the lactose permease of Escherichia coli.

Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.

Binding Sites↗

PubMed2004

Binding affinity of lactose permease is not altered by the H+ electrochemical gradient.

The x-ray structure of lactose permease of Escherichia coli (LacY) exhibits a single sugar-binding site at the apex of a hydrophilic cavity open to the cytoplasm, and it has been postulated that the binding site has alternating access to either side of the membrane during turnover. Here, the affinity of LacY for ligand in right-side-out or inside-out membrane vesicles is measured in the absence or presence of an H(+) electrochemical gradient (Deltamicro(H(+))) by utilizing ligand protection against alkylation. Right-side-out or inside-out membrane vesicles containing LacY with a single cysteine residue at position 148 exhibit K(D) values for lactose or beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside of approximately 1.0 mM or 40 microM, respectively, and no systematic change is observed in the presence of Deltamicro(H(+)) under conditions in which there is little or no accumulation of ligand. The results are consistent with a mechanism in which the major effect of Deltamicro(H(+)) on sugar accumulation is caused by an increased rate of deprotonation on the inner face of the membrane, leading to an increase in the rate of return of the unloaded symporter to the outer face of the membrane.

Alkylation↗

PubMed2003

Exploiting luminescence spectroscopy to elucidate the interaction between sugar and a tryptophan residue in the lactose permease of Escherichia coli.

The crystal structure of the Escherichia coli lactose permease at 3.5 A with a bound substrate has been reported recently. The structure reveals the sugar-protein contacts, which include hydrophobic stacking between the galactopyranosyl ring of substrate and the indole side chain of Trp-151, as proposed previously. The nature of this interaction is studied here by exploiting the luminescence properties of Trp-151 in a mutant devoid of other tryptophan residues. The following phenomena are observed. (i) The fluorescence emission spectrum of Trp-151 and fluorescence-quenching experiments with water-soluble quenchers demonstrate that Trp-151 is in a hydrophilic environment. (ii) Substrate binding leads to a blue shift in the emission spectrum and reduction in accessibility to polar quenchers, indicating that Trp-151 becomes less exposed to aqueous solvent. (iii) The phosphorescence spectrum of Trp-151 is red-shifted in the presence of substrate, indicating charge separation of the triplet state due to a direct stacking interaction between the galactopyranosyl and indole rings. The spectroscopic data fully complement the x-ray structure and demonstrate the feasibility of fluorescence spectroscopy for studying sugar-protein interactions.

Amino Acid Substitution↗

PubMed2003

Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli.

Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from the aqueous phase are susceptible to cross-linking. This approach is now extended to most of the remaining positions in the molecule. Of an additional 143 single-Cys mutants studied, homodimer formation is observed with both a 5-A- and a 21-A-long crosslinking agent containing bis-methane thiosulfonate reactive groups in 33 mutants and exclusively with the 21-A-long reagent in 43 mutants. Furthermore, intermolecular cross-linking has little or no effect on transport activity, thereby providing further support for the argument that lactose permease is functionally, as well as structurally, a monomer in the membrane. In addition, evidence is presented indicating that reentrance loops are unlikely in this polytopic membrane transport protein.

Amino Acid Sequence↗

PubMed2003

Aromatic stacking in the sugar binding site of the lactose permease.

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.

Alkylation↗

PubMed2002

Probing the mechanism of a membrane transport protein with affinity inactivators.

Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala(122)-->Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (Deltamu(H(+))) markedly increases the rate of reaction between Cys(122) and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala(122)-->Cys mutation is combined with a mutation (Cys(154)-->Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and Deltamu(H(+)) has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala(122) in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.

Affinity Labels↗

PubMed2002

An approach to membrane protein structure without crystals.

The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H(+) across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a related group of membrane proteins with 12 transmembrane domains that mediate transport of various substrates across cell membranes. Despite decades of effort and their functional importance in all kingdoms of life, no high-resolution structures have been solved for any member of this family. However, extensive biochemical, genetic, and biophysical studies on lactose permease have established its transmembrane topology, secondary structure, and numerous interhelical contacts. Here we demonstrate that this information is sufficient to calculate a structural model at the level of helix packing or better.

Binding Sites↗

PubMed2002

Changing the lactose permease of Escherichia coli into a galactose-specific symporter.

N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and beta,d-galactopyranosyl 1-thio-beta,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [(14)C]NEM. Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [(14)C]galactose transport by lactose, melibiose, or beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. In addition, although the conservative mutation Ala-122 --> Ser causes only mild inhibition of lactose transport, the mutations Ala-122 --> Phe and Ala-122 --> Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the non-galactosyl moiety of D-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides.

Alanine↗

PubMed2002

Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking.

Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant cross-linking of monomers to produce homodimers is observed in nine mutants with a 5-A-long cross-linking agent containing bis-methane thiosulfonate reactive groups [position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 263, 275, and 278 (helix VIII); and positions 308 (helix IX) and 398 (helix XII)]. The results are consistent with a current helix-packing model of the permease. Seven of the nine mutants that exhibit intermolecular cross-linking are located at or near the cytoplasmic ends of transmembrane helices; two are near periplasmic ends. The results suggest that only those Cys replacements accessible from the aqueous phase and not from the hydrophobic core of the membrane are susceptible to cross-linking because of the much higher reactivity of the thiolate anion relative to the thiol. Single-Cys mutants at positions 278 (helix VIII) and 398 (helix XII), which are located in opposite sides of the 12-helix bundle, exhibit similar rates of cross-linking with sigmoid kinetics. Furthermore, cross-linking is markedly decreased at 0 degrees C, suggesting that lateral diffusion of the permease within the plane of the membrane is important for intermolecular cross-linking. The findings confirm previous observations indicating that intermolecular cross-linking is a stochastic process resulting from random collisions and support a number of other lines of evidence that lactose permease is a monomer.

Amino Acid Sequence↗

PubMed2002

Helices VII and X in the lactose permease of Escherichia coli: proximity and ligand-induced distance changes.

By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N(7)/C(5) split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-specific cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A), N,N'-o- phenylenedimaleimide (6 A) and N,N'-p-phenylenedimaleimide (10 A). Mutant Asp240-->Cys (helix VII)/Lys319-->Cys (helix X) cross-links most efficiently with the 3 A reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H(+) symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H(+) from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).

Amino Acid Sequence↗