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THE LAC PERMEASE OF ESCHERICHIA COLI: SITE-DIRECTED MUTAGENESIS STUBIES ON THE MECHANISM OF ~-GALACTOSIDE/H ÷ SYMPORT P.D. Roepe, T.G. Consler, M.E. Meuezes and H.R. Kaback (*)

Department of Physiology, .~Ioward Hughes Medical Institute~Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90024-1570 (USA) Summary.

In this communication, we summarize site-directed mutagenesis studies of the lac permease from Escherichia coil, a prototypic H+-coupled active transport protein. We classify mutant permeases by phenotype, and suggest possible roles for some individual residues in the mechanism of H +/lactose symport. Although high-resolution structural information is not presently available, kinetic analysis of the partial reactions catalysed by the mutant permeases, as well as biophysical studies, suggest an evolving model for the mechanism of H +/lactose symport. Introduction.

The lac permease of E. coil is a polytopic inner membrane protein that catalyses the symport (i.e. co-transport) of one [3-galactoside molecule with one H + in response to a proton electrochemical potential (A12u +) (Kaback, 1987; 1989). Under physiologic conditions, where A~H+ is interior negative and alkaline, the permease couples the free energy released from the downhill translocation of H + to drive the accumulation of [~-galactosides against a concentration gradient. The permease also catalyses the thermodynamically equivalent converse reaction, performing uphin H + transport in response to downhill substrate translocation in either

direction across the membrane. Thus, lac permease is a model system for the study of free energy transduction performed by a host of biological machines. It is hoped, therefore, that the experimental approaches developed in the analysis of permease structure/function will aid the study of other membrane proteins and that the concepts which evolve will enhance our understanding of transport phenomena in general. Lac permease is encoded by the lacY gene, which has been cloned and sequenced (Biichel et al., 1980). The protein has been purified to homogeneity from the inner membrane, reconstituted into proteoliposomes, and shown to be solely responsible for lactose transport as a monomer (Newman et al., 1981 ; Viitanen et al., 1984, 1985; Costello et aL, 1987). Recently, Roepe and Kabaek (1989b) have described an alternative protocol for purification of the permease in a metastable water-soluble form based on overexpression of the lacy gene by the T7 RNA polymerase system of Tabor and Richardson (1985). Hydropathy analysis of the pr'.'mary sequence in concert with circular dichroism (CD) suggests that the protein is composed of twelve hydrophobic, membrane-spanning helices connected by hydrophilic "loops" with hydrophilic amino and carboxyl termini on the inner surface of the membrane. The salient features of this model are supported by chemical modification (Page and Rosenbnsch, 1988), limited pro-

(*) To whom correspondenceshould be adressed.

BACTERIAL teolysis (Goldkorn et aL, 1983; Stochaj et aL, 1986; Page and Rosenbusch, 1988), binding studies with site-directed polyclonal (Seckler et aL, 1983. 1986; Carrasco et al., 1984a; Seckler and Wright, 1984) and monocional antibodies (Carrasco et al., 1982, 1984b; Herzlinger et al., 1984, 1985), as well as Raman (Vogel et aL, 1985) and Fourier transform infrared (Roepe, P.D., Kaback, H.R. and Rothschild, K.J., unpublished data) spectroscopy. Most recently, J. Calamia and C. Manoil (1990) have provided strong support for more detailed aspects of the predicted topology by analysis of a series of lacY-phoA and lacY-lacZ fusions (Manoil and Beckwith, 1985). Finally, electron microscopy of freezefractured specimens reveals the presence of a notch or cleft within the permease (Li and Tooth, 1987; Costello et al., 1987). Application of oligonucleotide, sitedirected mutagenesis to the study of protein structure/function relationships has proven invaluable. Ideally, mutagenesis should be used in concert with crystallographic methods in order to obtain detailed structure/function information at the level of individual amino acid residues. Although memb r a n e proteins are d i f f i c u l t to crystallize, it has become apparent in the last few years that even in the absence of high-resolution structural information, important data can be obtained regarding those amino acid residues that are important for activity and membrane insertion. Moreover, testable models can be formulated which invite further investigation. Materials and methods. Materials.

1-14C-Lactose and p-nitro-3H-phe nyl-~,D-galactopyranoside were synthesized by Yu-Ying Liu under the direction of Arnold Liebman (Isotope Synthesi~ Group at Hoffmam~-La Roche, Inc.). All other materials were reagent grade and obtained from commercial sources.

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291

Methods.

Bacterial strains. - - The following strains of E. coli K-12 were used" J M I 0 1 , supE, thi, A(lacproAB), [F',traD36, p r o A B , laclqZAMl5] (Yanish-Perron et aL, 1985); T206 [lacI +O ÷ , Z - , Y - (A), rpsL, met-, thr-, recA, hsdM, hsdR/F', laclqO +ZAI I8(Y÷A+)] harbouring plasmid pGM21 [lacA(1)O+ P+ A(Z)Y+ A(A)tet r] (Teather et al., 1980); T!84 [T206 cured of plasmid pGM21 (Teather et al., 1980)]; HB 101, hsd s20 (r -B, m-a), rec AI3, ara-14, proA2, lacYl, gaIK2, rpsL20(Smr), xyl-5, mtl-l, supE-44, l - / F - (Boyer and Roulland-Dussoix, 1969); IlMH71-18 mutL [A(lacpro), SupE, thi/proA +B ÷, lac lq lacZ~MutL::TnlO] (Kramer et al., 1984); CJ236 dut, ung, thi, relA; pCJl05(Cmr) (Bio-Rad); MV! 190 A(lac proAB), thi, supE, Asrl- rec A)306::TnlO (tetr) iF': tra D36, pro AB, IaclqZAMIS] (Bio-Rad). Site-directed mutagenesis. - Oligonucleotide-directed, site-specific mutagenesis using M l 3 m p l 9 as the cloning vector was carried out either as described (Sarkar et aL, 1986) with given modifications, or by the uracil method of Kunkle (1985). Deoxyoligonucleotide primers complementary to antisense lacY DNA with the exception of one or two base mismatches were synthesized on an "Applied Biosystems" synthesizer and purified by polyacrylamide gel electrophoresis. Oligonucleotides were annealed to either thymidine- or uracil-containing singlestranded M l 3 m p l 9 template DNA harbouring the antisense fragment of lacY, and heteroduplex DNA was synthesized overnight at 14°C. In the former case, the repair-deficient strain E. coil BMH71-18 mutL (Kramer et al., 1984), was transformed with heteroduplex DNA in order to maximize the yield of mutant progeny. With the uracil method, E. coil M V l l 9 0 was transformed with heteroduplex DNA in order to destroy uracil-containing wildtype single-strand DNA. In some cases, colonies were screened by hybridization with appropriate 32p-labelled mutagenic primers, and all

292

6 t'~ F O R U M I N M I C R O B I O L O G Y

mutations were verified by dideoxynucleotide sequencing (Sarkar et aL, 1986; Sanger et aL, 1977). The replicative form of Ml 3 DNA containing specified mutations was isolated by alkaline lysis, restricted with EcoRI and the fragment containing lacY was cloned into the plasmid pACYC184. Orientation of l a c y in the recombinant plasmid was determined by Hinell restriction enzyme analysis (Sarkar et aL, 1986). In all cases where the activity of the mutated permease was compromised, the entire lacY gene was sequenced to insure that no secondary mutations contributed to the phenotype. In cases where the effect of more than one mutation was studied, mutagenesis was performed sequentially, using singlestranded DNA with one mutation as the template for a second mutation, and so on. E. coli HBI01(Z÷Y-) was transformed with plasmids encoding mutant lac permeases, and the cells were grown initially on eosin/methylene blue (EMB) plates containing 25 mM lactose as a qualitative estimate of permease activity (Miller, 1972). In addition, E. coil T 1 8 4 ( Z - Y - ) was transformed, and transport was measured quantitatively with l-R4C-lactose. Transport measurements. - - Active transport of lactose was measured using whole E. eoli T184 cells harbouring given plasmids as described (Piittner et aL, 1988), or by using E. coil right-sideout (RSO) vesicles in the presence of exogenous electron donors (Konings et al., 1971). Cells were grown at 37°C to an OD4z0 of 0.5, induced with 0.2 mM i-l: opyt-1 ~thio-13,D-galactopyranoside (1PTG) and grown for another 90 rain. Cells were harvested by centrifugation, washed extensively with 50 mM potassium phosphate (KPi; pH 7.5)/ 10 mM magnesium sulphate and resuspended in the same salt solution to an OD4z9 of 10.0 (approximately 1 mg of protem/ml). The cells were either used directly for transport (50 lzl-aliquots were incubated at room temperature, and IJ4C-lactose (10 mCi/mmol) was added to a final concentration of 0.4 raM), or vesicles were prepared as described by Kaback (1971) and assayed (50-1zl aliquots were incubated with the labelled lactose in the presence of

oxygen, ascorbate and phenazine methosulphate) (Konings et aL, 1971). At given times, the reactions were terminated by rapid dilution with 3.0 ml of 100 mM KPi (pH 5.5)/ 104)mM ~ithium chloride/20 mM mercuric chloride and immediate filtration through " W h a t m a n G F / F " glass fibre filters. Radioactivity retained on the filters was assayed by liquid scintillation spectrometry. Efflux and equilibrium exchange measurements were performed either with RSO vesicles prepared as described (Kaback, 197t) or with intact ceils grown as described above and treated with 2 mM ethylenediaminetetraacetic acid (EDTA) for 1 rain at 37°C (Leive, 1955). Vesicles or EDTA-treated cells were equilibrated overnight with 10 mM l-)4C-lactose (10 mCi/mmol) in the presence of 0.2 ~M carbonylcyanide-mfluorophenylhydrazone (FCCP). Aliquots (2 ~tl) of the suspensions were then diluted rapidly into a 100-fold excess of 50 mM KPi (pH 7.5)/10 mM magnesium sulphate without (efflux) or with 10 mM unlabelled lactose (equilibrium exchange). At specified times, the reactions were terminated and assayed as described above. I m m u n o b l o t analyses. - - Immunoblots were carried out as described (Carrasco et aL, 1982; Herzlinger et aL, 1985; Lolkema et aL, 1988) with monoclonal antibody 4AIOR and 12Sl. labelled protein A. Binding o f ZH-NPG. - - Binding of NPG was determined in RSO membrane vesicles using a modified version of the method of Hsu and Fox (1970) or by flow dialysis as described (Rudnick et eL, 1976). Purification o f lac permease. - Permease was purified from E. coil membranes as described (Viitanen et aL, 1986), Fluorescence spectroscopy.

--

Steady state fluorescence spectra of detergenfflipid-sotubilizedlae permease were obtained with a "Perkin-Elmer MPF-66" fluorometer interfaced to a "Perkin-Elmer 7500" data analysis terminal. In both cases, the protein con-

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TRANSPORT

centration was approximately 50 p.g/ml. Excitation was at 28.'; nm (bandwidth 20 rim), and the scan rate was 1 nm/s. Results.

Below we summarize data on lac permease mutants grouped according to a particular native amino acid residue (sections A-D), or according to functional significance (sections E, F). These data are then considered in their entirety in the "Discussion" to formulate a w o r k i n g model for H + / l a c t o s e symport. A) Cysteine

residues.

The early studies of Fox and Kennedy (1965), which d e m o n s t r a t e d substrate protection against N-ethylmaleimide (NEM) inactivation o f lac permease, suggested the involvement of an essential sulphydryl in or near the

293

binding site. Subsequently (Beyreuther et aL, 1981), the substrate-protectable Cys residue was identified as Cys148, predicted to be in putative helix V (fig 1) Based on these observations Trumble et al. (1984) and Viitanen ez aL (1985) converted Cys148 to Gly by oligonucleotide-directed, site-specific mutagenesis, and observed that C 148G (*) permease exhibits an initial rate of lactose transport that is about 25 % of the rate of the wild type, a n d a steady state level o f accumulation comparable to the wild type (table I). Interestingly, the mutant protein is ina c t i v a t e d b y e x p o s u r e to N E M , although the rate of inactivation is slower than that observed for wild-type permease. However, galactosyl I-thio[3-D-galactopyranoside (TDG) affords no protection against inactivation. Further studies (Neuhas et al., 1985 ; Sarkar el al., 1986a) demonstrate that C148S permease catalyses transport as well as the wild type and exhibits the same properties as C148G permease with regard

tnl o

~

Q

IN~ I K~S

o~,.

INHO

VT

QD

o~

_TI~

SAL

F

I

o

13

++

Op

NMy ~

FIG. 1. - - Model for the secondary structure oJ lac permease based on a variety Qf data. Roman numerals indicate numbering of helices and Arabic numerals indicate the numbering scheme for hydrophilic regions. The positions of Trp residues are highlighted.

(*) Site-directedmutants are designated as follows: the one-letter amino acid code is used followed by a number indicating the position of the residue in wild+typepermease. The sequenceis followed by a second letter denoting ;.he amino acid replacement at this position.

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OGY

TAaLE I. - - Summary of Cys mulations of lac permease.

Mutant C117S C148G C148S CI54G CI54S CI54V CI76S C234S C333S C353S/C355S

Initial rate of lactose transport (*) (% wild type)

Ref.

70 25 100 0 10 30 80 70 100 /> 50

Menick el al., 1987a. Trumble et aL, 1984. Sarkaf et al., 1986a. Menick et al., 1985. Menick et aL, 1985. Menick et al., 1987a. Brooker and Wilson, 1986. Brooker and Wilson, 1986. Menick et al., 1987a. Menick et al., 1987.

(*) Initial rate of transport is calculated from the linear portion of the uptake curve up to 15 s.

to NEM inactivation. Thus, although Cys148 is important for substrate protection against NEM inactivation, it does not play a direct role in the mechanism of lactose/H+ symport. Subsequently, site-directed mutagenesis of Cys154, predicted to lie near Cysl48 in the same helix, shows that this residue is important for transport (Menick et at., 1985). C154G permease exhibits no transport activity, while C154S permease and C154V permease catalyse transport at about 10 % and 30 % of the rate of wild-type permease, respectively (table 1). Moreover, all of the C154 mutants bind the high-affinity ligand p-nitrophenyl-ct,12galactopyranoside (NPG) normally. In addition, Brooker and Wilson (1986) substituted Set for Cys176 or Cys234, and Menick et al. (1987) replaced Cys117, Cys333 or Cvs353 and Cys355 with Ser, a n a a~[ o~ the }a;~tants exhibit significant transport activity (table l). The data taken as a whole demonstrate that of the eight Cys residues in the p~cmease, +only Cys154 is important for laetose/H symport, that this residue is not involved in either substrate binding or H + translocation, and that the electronegativity o f the residue at position 154 is not directly related to transport activity (Le., C154V permease is about 3-times more active than C154S

permease; table I). in light o f earlier suggestions that sulphydryl-disulphide interconversion might be important for transport activity (Kaback and Barnes, 1971; Konings and Robillard, 1982; Robillard and Koning% 1982), these studies clearly demonstrate the value of s i t e - d i r e c t e d m u t a g e n e s i s o f lac permease. Since C154 is the only cysteine residue that is important for activity, any postulated disulphide bond formation would be required to occur between permease monomers. However, the permease appears to be completely functional as a monomer (Costello et al., 1987), and it is functional even with Set or Val at position 154. It is highly unlikely, therefore, that sulphydryl/disulphide interconversion plays a role in the mechanism of lactose/H + symport. B) Tyrosine residues.

Since Tyr residues have been observed to protonate and deprotonate durin~ the H + - p u m p i n g photocycle o f bact e r i o r h o d o p s i n (Bogomolni, 1978; Rothschild et al., 1986; DoUinger et al., 1986; Roepe et al., 1987, 1988h) and are often components o f substrate-bindin~ sites (e.g. Wright, 1984), each of the 14 Tyr residues o f lac permease was

BACTERIAL replaced with Phe in order to assess the importance of the tyrosyl hydroxyl group for lactose/H + symport a n d / o r substrate recognition (Roepe a n d Kaback, 1989a). As summarized in table II, ten of the mutations have no significant effect on permease activity. O f the four that do alter activity, replacement of Tyr26 or Tyr336 with Phe inactivates all modc.s of lactose translocation, and the binding affinity o f the mutant permeases for N P G is markedly decreased (i.e., K o is increased from the wild-type value o f 22 ~M to greater than 750 ~tM and greater than 1 mM, respectively). In addition, Y336F permease ts inserted into the memurane less efficiently than the wild type, as judged by immunoblot experiments. Y236F permease catalyses equilibrium lactose exchange about 40 070as well as the wild type, a process which does not entail net H + translocation, but does not perform active transport or efflux, both o f which occur in symport with H +. Finally, Y382F permease catalyses exchange as well as the wild type, but exhibits low rates o f active transport a n d efflux without being uncoupled, suggesting that Tyr382 plays a role in

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the step corresponding to return of the unloaded permease (see below). In view of the potential H + transfer capabilities of Tyr residues, these data suggest that Tyr26, -336 and possibly Tyr236 may be important for coupling lactose a n d H ÷ translocation. However, Brooker and Wilson (1985) showed that a mutant containing Asn in place of Tyr236 catalyses active maltose transport, albeit at a slow rate. Thus, if Tyr236 is an essential component of a H ÷ transport pathway during lactose/H* symport, some other group must compensate for its loss during the active transport o f maltose. Additionally, it is apparent thut this residue, as well as Tyr26 a n d Tyr336, is important for high-affinity binding o f substrate (table ll), thereby suggesting t h a t residues which are important for translocation may also be components of the substrate recognition site(s). Such dn hypothesis is consistent with the idea that ligand binding contributes part of the initial activation energy required for substrate a n d / o r H ÷ translocation. Thus, conformational changes that may accompany the binding o f substrate could alter the electrostatic environment

TABLE 11. - - Summary of Tyr to Phe mutatinns of lac permease.

Mutant

Initial rate o f lactose transport

Exchange rate (% of wild type)

Efflux rate

Y2F Y3F YI9F Y26F Y75F YI01F YI13F Y228F Y236F Y263F Y336F Y350F Y373F Y382F

100 100 100 0 90 100 100 100 0 100 0 90 100 30

100 100 100 0 100 100 100 100 40 100 0 100 100 100

!00 100 100 0 > 90 100 100 100 0 100 0 > 90 > 90 30

Data taken from Roepe and Kaback, 1989a. ND = not done.

KD for N P G ND ND ND 750 ~M ND ND ND ND 170 p.M ND > lmM ND ND 100 ~M

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of groups in or near the binding site, resulting in their protonation/deprotonation and the initial " i n j e c t i o n " of a H + defect into the transport pathway (Nagle and Nagle, 1983; Nagle and Mille, 1981). Finally, it is noteworthy that of the ten non-essential Tyr residues identified in lac permease, only four are conserved in the sequence of the lac permease o f Klebsiella pneumcniae (McMorrow et al., 1988). Moreover, eaeil of the four essential Tyr residues is conserved.

C) Proline residues. A unique property of Pro residues is the "curling b a c k " o f its side chain in covalent linkage to the peptide bond nitrogen, thus forming a pyrrolidine ring which makes the peptide bond rigid. This has been hypothesized to be important for the formation of kinetic intermediates during protein folding (Brandts et aL, 1975) and has also been proposed to be a potential H 4 translocation mechanism, by providing a "torsionally-dependent" H + d o n o r / acceptor (Dunker, 1982). Additionally, hydropathy analysis of the lac permease predicts that most of the P r o residues are in intramembranous regions, that are likely to be helical based on lacYphoA fusions and spectroscopic data. This is not unique to lac permease, as Pro residues are predicted to lie within transmembran¢ helices of several other membrane proteins, including bacteriorhodopsin. Since statistical analysis of known protein structures predicts that Pro residues are unlikely to be in helical regions of proteins (Chou and Fasman, 1974), it has been suggested that structural discontinuities (i.e. " k i n k s " ) exist in the transmembrane helices of transport proteins and that these kinks are important for function a n d / o r membrane insertion. Von Heijne (1986) has pointed out that P r o residues are most frequently found in those helices which are oriented with their amino terminal " e n d s " pointed towards the extracellular face aild suggests that this disposition plays a role in determination of membrane protein topology.

This laboratory has investigated the role o f Pro residues in lac permease by systematically replacing each P r o residue with Gly, Ala, or Leu (Lolkema et al., 1988; T. Consler, O. Tsolas and H.R. Kaback, unpublished information). GIy, like Pro, is predicted to be a helix " b r e a k e r " , while Ala and Leu are helix " f o r m e r s " , and Gly, Ala and Leu are incapable of acting as a H + donor/acceptor. O f the 12 Pro residues in lac permease, none seem to be absolutely required for activity or insertion (table Ill; Overath et al., 1987; Roepe et aL, 1989), as they can be replaced by Gly or Ala or removed by truncation of the carboxyl-terminal tail o f the permease (Roepe et aL, 1989) and the resulting mutants retain some activity. Pro327 which has been studied in detail (Lolkema et al., 1988), can be replaced with Gly, Ala or Ser with little or no effect on lactose accumulation, while replacement with Thr or Cys results in low but significant activity and replacement with Leu, lie or Val completely abolishes activity. Although it is impossible to determine the role of Pro residues in the permease without a highresolution structure, it is apparent that the effects of the replacements at all but one position (i.e. 28) are due to specific chemical properties of the side chains (i.e., bulk, hydropathy a n d / o r ability to hydrogen bond). We cannot rule out at this time, however, the possibility that Pro28 may he acting as an important conformational determinant. In addition, the following possibilities merit consideration. 1) The Pro residues may be in helical domains, but cause little or no structural discontinuity. 2) The P r o residues may be in helical domains and cause structural discontinuities that are not essential for activity or insertion. 3) The Pro residues may be in nonhel;cal domains. In any event, the results highlight one of the caveats inherent in applying principles derived from statistical studies or, globular proteins to hydrophobic membrane proteins.

D) Tryptophan residues. The principal difficulty in the use of Trp fluorescence as a probe of protein

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297

TA•I.E II!. - - Summary of Pro mutations in lac permease.

Residue 28 31 61 89 97 123 192 220 280 327 403 (truncation) 405 (truncation)

Initial rate of lactose transport for substitution to: Gly Ala Leu (07o wild type) 15 90 82 96 90 100 86 94 68 12 ---

15 64 73 100 98 100 80 93 ~'~ 9"1 ---

26 14 97 75 84 17 ND 100 !2 0 100 100

Ref. T. Consler et al., unpublished " " " " " " " Lnlkema et hi., 1988 Roepe et al., 1989 "

Although the initial rate is impaired in P327G permease,a normal steady-statelevel of accumulation is observed. ND = not done.

structure/function is the complexity of the fluorescence signal, a consequence of the presence of multiple Trp residues, particularly in larger proteins. In order to circumvent this dilema, lac permease has been engineered such that the six native Trp residues are simultaneously replaced by Phe. The " T r p - l e s s " permease (W6F) retains at least 70 % of the transport activity of wild-type permease (Menezes et al., 1990) thereby facilitating detailed fluorescence studies of permease molecules with Trp residues introduced at specified positions by means of site-directed mutagenesis. The approach shoald be valuable for studying topology (through the analysis of fluorescence emission maxima ~nd accessibility to quenchers), and conforma*ional changes that may accompany the catalytic cycle, as well as the localization of the substrate-binding site. For example, recent measurements indicate that about 15 % of the Trp fluorescence intensity of lac permease is quenched on addition of [3,D-galactopyranosyl 1-t hio-[3-D-galactopyranoside (TDG) (M.E. Menezes, P.D. Roepe and H.R. Kaback, unpublished data). Furthermore, preliminary data imply the phenomenon in~'.~lves as many as five of the six native Trp residues, suggesting

that rather large scale environmental alterations involving helices 1-6 accompany binding of substrate. Raman spectroscopy also suggests that Trp residues are perturbed during association o f ligand (V6gel et al., 1984) as peaks near frequencies expected for Trp-ring vibrations (Roepe et al., 1988b) are present in R a m a n difference spectra. Relatedly, preliminary Fourier transform infrared difference data (Roepe, P.D., Kaback, H.R. and Rothschild, K.J., unpublished) suggest that the secondary structural alterations that accompany substrate binding are minimal and do not involve helical regions o f the protein. Thus, any model for binding should include small changes in secondary structure accompanied by relatively large tertiary structural alterations. Figure 2 shows the steady state fluorescence emission spectrum of detergent/lipid-solubilized wildqype and W6F permeases (Menezes et al., 1990). The W6F construct loses at least 80 070 of tae fluorescence intensity attributed to Trp residues. Residual intensity near 350 nm is likely due to protein contamination in the added lipid. Interestingly, increased intensity is observed at a wavelength expected for Tyr t?uorescence (i.e., 306 nm) most likely

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..~--,=

300

I 320

I 340

I 360

I 360

400

Wavelength(nml FIG. 2. -- Steady state fluorescence of lipid/detergent-so/ubilized wiM type and W6Fpermease. Note the drastic decrease in intensity at wavelengths expected for Trp in spectra of the W~F construct, as well as the increase at 306 nm due to increased Tyr fluorescence.

due to elimination of energy transfer to Trp residues in the W6F construct. Thus, the construct should also be valuable for probing the fluorescence properties of Tyr residues. Currently, studies are underway to determine the feasibility of monitoring the fluorescence signal from individual Trp residues introduced at novel locations by dsing the W6F construct as a template for further mutagenesis. E) Putative binding-site residues. Quiocho and coworkers have recently crystallized the periplasmie arabinosebinding protein (ABP) (Quiocho and Vyas, 1984) and the galactose-binding protein (GBP) (Vyas et al., 1988) from E. coil in the presence of substrate, and have, refined the structures to 1.7 and 1.9A resolution, respectively, thereby enabling identification of residues in-

timately involved in coordination of sugars to these enzymes. Both ABP and GBP are known to bind galactose. Since lactose is a [3-galactopyranoside and lac permease transports and binds galactose, we have compared the sequences of the three proteins in order to identify residues which might form part of the galactoside-binding domain of lac permease. Although overall sequence homology between the three proteins is poor, comparison of the ABP and GBP residues involved in sugar binding reveals several distinct similarities. Most of the residues involved in coordinating ligand are scattered throughout the primary sequences; however, both binding sites include as coordinating groups two residues within a contiguous fiveresidue sequence. This sequence ineludes a positively charged Arg or Lys, followed by a residue capable of forming a strong hydrogen bond, followed after two intervening residues by a

BACTERIAL TRANSPORT negatively charged Glu or Asp. The arabinose-binding site ~n ABP also contains two additional residues in a different contiguous five-residue sequence, Ar~ 151 and Thr 147, which are involved m hydrogen bond formation to the r:~ng oxygen and the C4 anomeric hydroxyl. Perusal of the lac permease sequence reveals that four five-residue sequences of the first type described abo,,e exist in the protein. However, one is in the carboxyl terminus, which is unimportant for function (Roepe etaL, 1989). The hydrophobicity profile of the protein predicts that two of the remaining sequences (EI30-RI34 and RI35-EI39) are disposed towards the cytoplasmic face of the membrane, while one (R255-E259) is disposed towards the extrafacial surface. Only two five-residue sequences not in the carboxyl terminus that contain a positive charge and an h y d r o x y l - e o n t a i n i n g residue four residues away exist in the sequence of the permease. One (K335-$339) is disposed towards the ~-ntrafacial surface, and the other (R302-$306) is in putative helix IX, We have subjected the positively and negatively charged residues, as well as the hydroxyl-containing residues, of these sequences to site-directed mutageaesis (Roepe, P.D.. Menezes, M.E. and Kaback, H.R., in preparation; Menick etal., 1987b) (table IV). Relatedly, Brooker and coworkers have screened for lac permease mutants with altered substrate specificity (Collins et aL, 1989; Franco etal., 1989) and find several mutations involving residues within these sequences (see below). In additi~)n, since the binding sites of the two crystallized proteins also contain a number of Asn and additional charged residues, we have mutated similar residues in lac permease predicted to be disposed to the cytoplasmic face of the membrane (fig. I and table V). In some cases, two mutations at a particular site were made, one which could perhaps conserve H-bonding capability of tlte residue, and another that would be expected to destroy it. Interestingly, mutation of the charged residues in any of the five-residue so-

299

quences described above results in significant loss of activity for at least one mutation, with the most dramatic effects occurring in sequences 4 and 5, the sequences not containing a negative charge (table IV). Mutation of E255 to Q, R259 to H and $339 to C results in mutant permeases with near wild-type levels of activity; however, when mutations were made at these positions such that H-bondlng capability is lost, 75 %, 25 % or 50 % loss of transport activity, respectively, is observed. Thus, even in the absence of direct binding data for these mutants, the transport data are consistent with the idea that each of these sequences is important for activity, possibly because they are involved in substrate recognition, tt is noteworthy that the R302-$306 segment mentioned above is the only sequence predicted to reside within a transmembrane domain. This suggests that at least part of a binding site is hydrophobic, a conclusion consistent with a Variety of other data (Sehuldiner etaL, 1976; Goldkorn etaL, 1983). Mutation of each charged residue not in the five-residue sequences and each Asn predicted to be disposed to the extrafacial surface of the membrane are summarized in table V(fig. 1). Residues which appear to be most important for activity are clustered in hydrophilic segments 2, 8 and 12 (fig. 1) which compose the largest extrafacial loops of the permease. The observations of Overath and coworkers (1987) and Brooker and Wilson (1985) are consistet~t with the hypothesis that hydrophUic sequents 2 and 8 comprise part of a substrate-binding site. These workers described mutations in and near these regions which alter substrate affinity. The data obtained for sequence 3 (i.e. hydrophilic segment X, table IV) is also consistant with the notion.

F) Arg302, HIS322 and Glu325 as c o m ponents o f a possible H + relay.

Chemical modification studies with diethylpyrocarbonate or rose bengal provided an initial clue that His residues are important in coupling H + and lac-

300

6 th F O R U M I N M I C R O B I O L O G Y

TABLE IV. - - Summary of mutations of residues within contiguous five.residue sequences exhibiting homology to sequences in the binding domain of the ABP or GBP proteins. Mutant

Initial rate of active lactose transport (% wild type)

Sequence 1 (disposed to cytoplasm) E 130Q S133C S133A R134N RI34E

30 100 80 30 10

Sequence 2 (disposed to cytoplasm) R135N S136C SI36A E139Q

30 100 100 25

Sequence 3 (disposed to periplasm) E255Q E255V T258V R259H R259L

100 25 0 100 75

Sequence 4 (helix IX) R302L R302H R302Q $306A (*) $306T (*) $306L

0 0 0 I00

Sequence 5 tdisposed to cytoplasm) K335L Y336F $339C $339A

0 0 90 50

(*) Since active lactose transport data are not availablefor these mutants, tee reader is referredto Collins et al.. 1989 for a discussion of the effects of these mutations. See figure I for the location of each sequence.

tose translocation (Padan et al., 1979; Garcia et al., 1982), and subs~.queutly, each of the four His residues in lac permease was changed to Are, Asn, Gin or Lys (Padan et al., i985; Piiltner et al., 1986, 1989). Replacement of His35 and His39 with Arg or replacement of His205 with Arg, Asn or Gin has no effect on H*/lactose symport, while replacement of His322 with Arg, Asn,

Gin or Lys results in dramatic loss of activity. Strikingly, however, H322R permease catalyses downhill lactose influx at high external lactose concentrations, without c o n c o m i t a n t H + translocation (i.e., thepermease is uncoupled) (Piittner et al., 1989). In view of these observations, attention was focused on Glu325, which is predicted to lie on the same face of

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301

TABLE V . - - Sr:.nmary of mutations of Ash residues and charged residues predicled to be disposed on the periplasmic surface of lac Bermease (fig. 1).

Mutant

Initial rate of active lactose transport (% wild type)

H35R D36N N381 H39R K42L D44N NI021 N1631 N1641 D237N D240N N2451 E314Q N371 i E374Q

100 0 30 100 0 100 50 100 10 15 10 100 100 50 0

helix X as His322 (fig. I). Molecular modeling suggests that the earboxylatg of Glu325 should be within about 1.5 A of the imidazole group o f His322, well placed for formation of an H bond. Permease with Ala, Gin, Val, His, Cys or Trp in place of Glu325, like the His322 mutants, does not ¢atalyse either active transport or efflux (Carrasco et al., 1986, 1989). Remarkably, however, each mutant catalyses equilibrium lactose exchange as well as the wild type. Moreover, permease mutated at position 325 catalyses eounterflow at the same rate and to the same extent as wild-type permease, but the internal concentration of 14C-lactose is mainrained for a prolonged period due to the defect in efflux. These data can be rationalized by the simple kinetic scheme presented in figure 3. Efflux down a concentration gradient consists of a minimum of five steps: (1) binding of substrate and H + on the inner surface o f the membrane (order unspecified); (2) translocation of the ternary complex to the outer surface; (3) release of substrate; (4) release

of H + ; and (5) return of the unloaded permease to the inner surface. Alternatively, exchange and eounterflow with external lactose at saturating concentrations involves steps 1-3 only. Notably, release of H + (step 4) appears to be rate-limiting for the overall cycle (Kaczorowski and Kabaek, 1979), and since replacement of Glu325 results in a permease that is defective in all steps involving net H + translocation but catalyses exchange and counterflow normally, these mutants are probably blocked at step 4 (i.e., they are unable to lose H+). The His322 mutants, on the other hand, may be blocked in the initial protonation step. When Glu325 is replaced by Asp, the protein retains about 20 % of the transport activity of wild-type permease (Carrasco et aL, 1989; P.D. Roepe et al., m a n u s c r i p t in p r e p a r a t i o n ) , demonstrating that an acidic residue at position 325 is critical for lactose/H + symport. The E325D mutant, however, exhibits some additional, remarkable characteristics (P.D. Roepe et al.; P.D. Roepe and H.R. Kaback,

302

6 "~ F O R U M I N M I C R O B I O L O G Y

IN

OUT

Hj

8~

HI ;C

~ C~H

FIG. 3. - - Schematic representation of reactions involved in efflux, exchange and counterflow. C represents permease; S is substrate (lactose). The order of substrate and H + bir~fiingat the inner surface of the membrane is not implied (from Kaczorowski and Kaback, 1979).

manuscripts in preparation). Figure 4 shows the initial rate of lactose transport as a function of bulk pH for vesicles containing.either wild-type or E325D permease m the presence or absence of an applied pH gradient (ApH) formed by diffusion of acetate out of RSO vesicles (i.e., interior alkaline). The initial rate of active transport catalysed by E325.'3 permease is stimulated six-fold at physiological pH upon imposition of the ApH, while no stimulation is observed for the wild type, presumably because it is already functioning at optimal efficiency. The results are strong support for the notion that H + transfer involving the acidic residue at position 325 is a key step in H , / l a c t o s e symport. S u r p r i s i n g l y , E325D p e r m e a s e catalyses equilibrium lactose exchange in a highly pH-dependent manner (fig. 5). Thus, at pH ~< 7.7, the mutant performs exchange at rates similar to the wild type, but at higher pH, exchange is progressively and reversibly inhibited with a midpoint at about pH 8.5. Notably, the wild-type protein

exhibits no sensitivity to pH in similar measurements. Since molecular modeling suggests that the carboxylate at 325 and the His322 imidizole are close enough to hydrogenhond, and since replacement of Glu325 by Asp would mcrease the distance between these groups, thereby weakening an H bond between the two groups, the data suggest that translocation of the loaded permease does not tolerate the presence of a negative charge at position 325 and that the carboxylate at position 325 undergoes protonation/deprotonation during symport. Although the exchange data also imply that Asp325 may have an anomalously high PKa, it should be noted that a negatively charged Asp residue which protonates during the H + pumping photocycle of bacterior h o d o p s i n has been observed at pH >I 8.5 (Roepe et al., 1987). Alt~ernatively, it is also possible that the apparent pK a reflects a perturbed His residue at position 322. Based on the results for the His322 and Glu325 mutants, it has been proposed that the two residues may be corn-

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303

TRANSPORT

,4t 1.2

~ c:, 0 . 8 .,=

~g

o.6

o.4 0.2

O.C

I 5

J

I 8

f

I 7

,

I 8

I g

10

pH

FIG. 4. - - Initial rate o f &W-driven active lactose transport in E. coli vesicles harbouring either the wild-type or E325D permease in the absence or presence o f an artificially high ApH. Vesicles were equilibrated with 2 t.tM nigericin and 100 mM KPi at the indicated pH. Membrane potential was generated by the addition of aesorbate and phenazine methosulphate in the presence of oxygen as described (Kabaek, 1971), and ApH (interior alkaline) was generated by diffusion of 100 mM acetate out of the vesicles. 14C-lactose was added to a final concentration of 0.4 mM, and the reactions quenched by the addition of 100 mM KPi (pH 5.5)/100 mM LiCl/10 mM HgCl,. Radioactivity incorporated into the vesicles was determined by filtration through glass ~microfibre filters (Whatman, type GC/F) and liquid scintillation spectrometry. Initial rates were determined from the linear portion of the uptake curve.

ponents of an H + relay that is an important elem;nt of :he mechanism of coupling betwe:n H + and lactose translocation (Carrasco et al., 1986; Ptittner et al., 1986; Kaback, 1987). Attempts to pinpoint a Ser residue in the vicinity of His322 and Glu325 that might form another component of a charge relay, in analogy to the serine proteases led to replacement of Ser30~ (helix IX) or Ser306 with Ala. These mutations have no apparent effect on activity. Recently, Collins et al. (1989) have found that mutation of Ser306 to Thr or Leu results in a permease molecule with altered suhstrate specificity. Thus, $306T and $306L permease transport maltose (an ~-glucoside) significantly better than the wild type.

However, replacement of Arg302 with Leu, His or Lys yields permease with properties similar to those of permease harbouring mutations at His322, indicating that Arg302 may also be involved in the pathway of lactose-coupled H ÷ translocation (Menick et al., 1987b). Molecular modeling of putative helices IX and X suggests that the guanidinio group of Arg302 may be sufficiently close to His322 to form an H bond with the imidizole of His322, which in turn may be H-bonded to Glu325 Although it has been reported that replacement of Lys319 (helix X, three residues below His322) with Leu results in a permease with wild-type activity (Menick et al., 1987b), more recent studies have shown this conclusion

304

6 th F O R U M I N M I C R O B I O L O G Y

100

8O = o

60

•'~

40

./

2~

~ l l l l l l l l ~ 6

$

10

I

12

pH FIG. 5. - - Halftimes of equilibrium lactose exchange at various values of the bulk pH for wild-t))ve and E325D permease. E. coil vesicles were incubatedt4with 100 mM KPi at the "mdlcated'pH, 20 itM valinomycin, 2 ~tM n geric n and 10 mM C.lactose for 12 h at 4°C. Aliquots (2-~.1)were diluted rapidly into a 100-fold excess of buffer at the same pH containing equimolar unlabelled lactose, and the reactions were quenched as for figure 3. Also shown are data points obtained for wild-type vesicles which have been incapacitated in exchange by treatment with p-cbloromercaribenzenesulphonic acid (PCMBS).

to be incorrect. K319L permease manifests characteristics similar to E325A pernlease, suggesting that the positive c h a r g e o n Lys319 m a y modulate the H + transfer capabilities of nearby residues or that it may also be directly involved in H + translocation. In any case, it is clear that a cluster of amino acids within putative helices IX and X are highly important for both ligand-binding and H + / l a c t o s e symport. Indeed, binding studies with the high-affinity ligand N P G demonstrate that the His322 and Arg302 mutants exhibit drastically lowered affinities for the ligand. Conversely, the Glu325 mutants bind NPG with near wild-type affinity. Although E325D permease binds substrate with only a slightly elevated KD at pH 5.0 to p H 7.5, at high p H , binding is severely and pro-

gressively inhibited, suggesting that the introduction of a negative charge at p o s i t i o n 325 not o n l y d i s r u p t s translocation of the ternary complex, but lactose binding as well. Taken as a whole, these data suggest that the pathways for H ÷ and lactose may overlap (i.e. Arg302, His322 a n d Glu325 may reside in or near the substrate-binding site) and that protonation o f His322 a n d / o r Glu325 may be required for high-affinity binding. This hypothesis is conmstent with the existence of a strong H-bond network between Arg302, His322 and Glu325 in the wild-type protein. Perturbations of the PKa of the groups composing this Hbond network (i.e. the Glu325 to Asp substitution) would therefore be expected to result in new a n d / o r altered p H sensitivities to binding a n d / o r lactose translocation.

BACTERIAL

In any event, if these three residues are sufficiently close to H bond and function as a charge relay, the polarity, distance and orientation of the three residues, as well as their PKa, should be critical (Lee et al., 1989). The importance of polarity between His322 and Glu325 has been studied by interchanging the positions of the residues, and the resultant permease is inactive in all modes of translocatlon. The effect of distance and orientation between the two groups was studied by interchanging Glu325 with Va1326, therebyomoving the carboxylate about 1.5 A. This modified permease is also completely inactive, and introduction of a His residue at position 323 in this mutant in order to restore the wild-type o r i e n t d o n between the His and Glu does not restore activity, consistent with the contention that rotation of the two groups by 100° relative to Arg302 disrupts the H-bond network between the three residues. Discussion. The d a t a presented in this manuscript, taken in its entirety, suggest several key elements of the mechanism of H +/lactose active transport catalysed by the lac permease. l) D i s u l p h i d e / s u l p h y d r y l interconversion is not an integral part of the mechanism. 2) The pathways for H ÷ and lactose transloeation may share common residues, in particular those postulated to form a H + relay. 3) Association of ligand probably involves large-scale e n v i r o n m e n t a l changes with small secondary structural alterations. 4) The pathway for lactose may involve "binding sites" that include hydrophilic segments 2, 8 and 12 on the periplasmic surface, residues within putative helices IX and X, and hydrophilic segments 5 and l 1 on the cytoplasmic surface (fig. 1). Furthermore, the data in their entirety suggest possible arrangements of the 12 transmembrane helices in the permease, whereby key regions of the

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305

protein containing residues vital for various aspects of function are within reasonable proximity. One possible arrangement is shown below (fig. 6), although others can be postulated. The suggested arrangement is consistent with the physical size and shape of lac permease particles derived from freezefracture electron microscopy of :aroteoliposomes (Costello et al., 1987). It is proposed that helices I, V and X form an mner three-helix core in the molecule, thus placing in proximity residues in helix i (including Tyr26), helix V (including Cys154), and helix X (including His322 and Glu325) which have been shown to be important for active transport (see above). The arrangement also allows for interaction of residues in helices IX (A,g302) and X. It is envisioned that an " o p e n " and "closed" state for this arrangement exists (compare A and B) corresponding to translocation competent vs incompetent forms of the enzyme, respectively. Furthermore, the translocation competent structure is assumed to form after ligand is associated. Available data suggest that little change in secondary structure occurs during binding of ligand, however, environmental alterations near six different Trp residues on helices I, Ill, V and Vl presumably occur. Thus, a simple model for binding would entail formation of a channel during association of ligand which involves movement near Trp-containing helices such that the overall secondary structure remains relatively constant. The proposed arrangement also allows for hydrophilic segments 2, 8 and 12 (fig. I) to be in close proximity in the "closed" state such that a binding pocket containing residues in these segments as well as residues within helices IX and X would be possible. Also, hydrophilic segments 5 and 11, connecting helices IV/V and X/X1, respectively, are placed near each other in the " o p e n " state in the mouth of the pore such that residues in these regions could interact to form a site mediating the release of substrate on the cytoplasmic surface. It is envisioned that in the "closed" state, the extracellular binding site involving segments 2, 8 and 12 and residues in helices IX and X is in

306

6 tk F O R U M I N M I C R O B I O L O G Y

I

S.lnm

I

-00

%IsN A closed

'q.tsN

~ ................

Intrafacial extrafacial

g open

FIG. 6. - - Proposed geometric arrangement of the twelve helices of lac permease based on consideration of the mutagenesis work summarized in this manuscript. A = Closed state in which ligand is not associated; B = open state formed after association of ligand. Note the accessibility of helices I, V and X, as well as hydrophilic segments 2, 8 and 12 from the periplasmic surface and hydrophilic segments 5 and 11 from the cytoplasmic surface. Formation of the open state is envisioned to involve little secondary structural alteration, but relatively large scale environmental changes due to movement of helices I-IV relative to helices V-Xli.

a high-affinity conformation and the int r a c e l l u l a r b i n d i n g site involving segments 5 and I I is in a low-affinity conformation, and that the converse holds in the " o p e n " state. The suggestion that residues within helices 1X and X are involved in binding is consistent with the existence of a cleft in lac permease, which has been observed via electron microscopy. A simple scenario that would allow for the above involves binding-induced conformational changes which alter the affinities of the two sites. Although binding studies under deenergized conditions have not clearly resolved two distinct binding sites, it is possible that the intrafacial site is converted to a highaffinity c o n f o r m a t i o n only in the presence of a membrane potential, or during lactose translocation. In any case, it is proposed that binding induces protonation/deprotonation reactions of residues within or near the extrafacial site (such as Glu325 or His322) which propagate conformational alterations

via migration of an ionic (i.e. H +) defect from the extrafacial site to the intrafacial site. This hypothesis is consistent with the observed behaviour of permease harbouring GIu325 or His322 mutations. E325D permease is highly pH-dependent with regard to binding of substrate under deenergized conditions, and E325A permease is uncoupled, suggesting that the residue may be involved both in binding anti H + translocation. H322R permease has a lowered affinity for substrate and is similary uncoupled, performing downhill lactose influx without concomitant H + translocation. Thus, the present data suggest that the binding site for substrate on the extrafacial surface as well as the pathway for H + translocation overlap. The above model is only suggested in order to stimulate further investigation. Future site-directed mutagenests as well as biophysical studies will be needed to define the exact mechanism of H +/lactose symport.

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307

References.

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