Biomedical subjects

H Ronald Kaback

Publications and source records attributed to H Ronald Kaback.

At least 19 recordsLinked to original sources

PubMed2006

Site-directed alkylation and the alternating access model for LacY.

In a functional lactose permease mutant from Escherichia coli (LacY) devoid of native Cys residues, almost every residue was replaced individually with Cys and tested for reactivity with the permeant alkylating agent N-ethylmaleimide in right-side-out membrane vesicles. Here we present the results in the context of the crystal structure of LacY. Engineered Cys replacements located near or within the inward-facing hydrophilic cavity or at other solvent-accessible positions in LacY react well with this alkylating agent. Cys residues facing the low dielectric of the membrane or located in tightly packed regions of the structure react poorly. Remarkably, in the presence of ligand, increased reactivity is observed with Cys replacements located predominantly on the periplasmic side of the sugar-binding site. In contrast, other Cys replacements largely on the cytoplasmic side of the binding site exhibit decreased reactivity. Furthermore, both sets of Cys replacements in the putative cavities are located at the periplasmic (increased reactivity) and cytoplasmic (decreased reactivity) ends of the same helices and distributed in a pseudosymmetrical manner. The results are consistent with a model in which the single sugar-binding site in the approximate middle of the molecule is alternately exposed to either side of the membrane due to opening and closing of cytoplasmic and periplasmic hydrophilic cavities.

Alkylating Agents↗

PubMed2006

Direct sugar binding to LacY measured by resonance energy transfer.

Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for Förster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the Förster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.

Amino Acid Substitution↗

PubMed2006

Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli.

Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.

Calorimetry↗

PubMed2006

Site-directed alkylation of cysteine replacements in the lactose permease of Escherichia coli: helices I, III, VI, and XI.

To complete a study on site-directed alkylation of Cys replacements in the lactose permease of Escherichia coli (LacY), the reactivity of single-Cys mutants in helices I, III, VI, and XI, as well as some of the adjoining loops, with N-[14C]ethylmaleimide (NEM) or methanethiosulfonate ethylsulfonate (MTSES) was studied in right-side-out membrane vesicles. With the exception of several positions in the middle of helix I, which either face the bilayer or are in close proximity to other helices, the remaining Cys replacements react with the membrane-permeant alkylating agent NEM. In helices III and XI, most Cys replacements are also alkylated by NEM except for positions that face the bilayer. The reactivity of Cys replacements in helix VI is noticeably lower and only 45% of the replacements label. Binding of sugar leads to significant increases in the reactivity of Cys residues that are located primarily at the same level as the sugar-binding site or in the periplasmic half of each helix. Remarkably, studies with small, impermeant MTSES show that single-Cys replacements in the cytoplasmic portions of helices I and XI, which line the inward-facing cavity, are accessible to solvent from the periplasmic surface of the membrane. Moreover, addition of ligand results in increased accessibility of Cys residues to the aqueous milieu in the periplasmic region of the helices, which may reflect structural rearrangements leading to opening of an outward-facing cavity. The findings are consistent with the X-ray structure of LacY and with the alternating access model [Abramson, J., Smirnova, I., et al. (2003) Science 301, 610-615].

Alkylation↗

PubMed2006

Sequence alignment and homology threading reveals prokaryotic and eukaryotic proteins similar to lactose permease.

Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H(+) translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H(+) translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E.coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H(+) translocation in LacY.

Amino Acid Sequence↗

PubMed2006

Conservation of residues involved in sugar/H(+) symport by the sucrose permease of Escherichia coli relative to lactose permease.

Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB. Moreover, despite only 28% sequence identity and a marked difference in substrate specificity, the structural organization of the residues involved in sugar-binding and H(+) translocation is conserved in CscB. Functional analyses of mutants in the homologous key residues provide strong evidence that they play a similar critical role in the mechanisms of CscB and LacY.

Amino Acid Sequence↗

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

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↗

PubMed2005

Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli.

A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.

Amino Acid Substitution↗

PubMed2005

Asymmetric binding of membrane proteins to GroEL.

The interaction of GroEL with non-native soluble proteins has been studied intensively and structure-function relationships have been established in considerable detail. Recently, we found that GroEL is also able to bind membrane proteins in the absence of detergents and deliver them to liposomes in a biologically active state. Here, we report that three well-studied membrane proteins (bacteriorhodopsin, LacY, and the bacteriophage lambda holin) bind asymmetrically to tetradecameric GroEL. Each of the membrane proteins was visualized in one of the center cavities of GroEL using single particle analysis.

Bacteriorhodopsins↗

PubMed2005

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential.

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.

Adenosine Triphosphatases↗

PubMed2005

Structure and mechanism of the lactose permease.

More than 20% of the genes sequenced thus far appear to encode polytopic transmembrane proteins involved in a multitude of critical functions, particularly energy and signal transduction. Many are important with regard to human disease (e.g., depression, diabetes, drug resistance), and many drugs are targeted to membrane transport proteins (e.g., fluoxetine and omeprazole). However, the number of crystal structures of membrane proteins, especially ion-coupled transporters, is very limited. Recently, an inward-facing conformer of the Escherichia coli lactose permease (LacY), a paradigm for the Major Facilitator Superfamily, which contains almost 4000 members, was solved at about 3.5 A in collaboration with Jeff Abramson and So Iwata at Imperial College London. This intensively studied membrane transport protein is composed of two pseudo-symmetrical 6-helix bundles with a large internal cavity containing bound sugar and open to the cytoplasm only. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism is proposed in which the binding site is alternatively accessible to either side of the membrane.

Amino Acid Sequence↗

PubMed2004

Exploring the role of a unique carboxyl residue in EmrE by mass spectrometry.

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.

Antiporters↗

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↗

PubMed2004

Monitoring conformational rearrangements in the substrate-binding site of a membrane transport protein by mass spectrometry.

Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low ( approximately 25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding.

Arginine↗

PubMed2004

Role of YidC in folding of polytopic membrane proteins.

YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding. Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY. By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes. Moreover, LacY also folds improperly in proteoliposomes prepared without YidC. However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly. The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

Antibodies, Monoclonal↗