Early inherited risk for anxiety moderates the association between fathers' child-centered parenting and early social inhibition.

Studies of the role of the early environment in shaping children's risk for anxiety problems have produced mixed results. It is possible that inconsistencies in previous findings result from a lack of consideration of a putative role for inherited influences moderators on the impact of early experiences. Early inherited influences not only contribute to vulnerabilities for anxiety problems throughout the lifespan, but can also modulate the ways that the early environment impacts child outcomes. In the current study, we tested the effects of child-centered parenting behaviors on putative anxiety risk in young children who differed in levels of inherited vulnerability. We tested this using a parent-offspring adoption design and a sample in which risk for anxiety problems and parenting behaviors were assessed in both mothers and fathers. Inherited influences on anxiety problems were assessed as anxiety symptoms in biological parents. Child-centered parenting was observed in adoptive mothers and fathers when children were 9 months old. Social inhibition, an early temperament marker of anxiety risk, was observed at child ages 9 and 18 months. Inherited influences on anxiety problems moderated the link between paternal child-centered parenting during infancy and social inhibition in toddlerhood. For children whose birth parents reported high levels of anxiety symptoms, greater child-centered parenting in adoptive fathers was related to greater social inhibition 9 months later. For children whose birth parents reported low levels of anxiety symptoms, greater child-centered parenting in adoptive fathers was related to less social inhibition across the same period.

Control of H+/lactose coupling by ionic interactions in the lactose permease of Escherichia coli.

A combinatorial approach was used to study putative interactions among six ionizable residues (Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325) in the lactose permease. Neutral mutations were made involving five ion pairs that had not been previously studied. Double mutants, R302L/E325Q and D240N/H322Q, had moderate levels of downhill [(14)C]-lactose transport. Mutants in which only one of these six residues was left unchanged (pentuple mutants) were also made. A Pent269(-) mutant (in which only Glu-269 remains) catalyzed a moderate level of downhill lactose transport. Pent240(-) and Pent 322(+) also showed low levels of downhill lactose transport. Additionally, a Pent240(-) mutant exhibited proton transport upon addition of melibiose, but not lactose. This striking result demonstrates that neutralization of up to five residues of the lactose permease does not abolish proton transport. A mutant with neutral replacements at six ionic residues (hextuple mutant) had low levels of downhill lactose transport, but no uphill accumulation or proton transport. Since none of the mutants in this study catalyzes active accumulation of lactose, this is consistent with other reports that have shown that each residue is essential for proper coupling. Nevertheless, none of the six ionizable residues is individually required for substrate-induced proton cotransport. These results suggest that the H(+) binding domain may be elsewhere in the permease or that cation binding may involve a flexible network of charged residues.

Eosinophil activation and cysteinyl leukotriene production in infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: It has been suggested that acute infantile bronchiolitis associated with respiratory syncytial virus (RSV) may share some pathogenic features with atopic asthma in that virus-specific IgE is produced and cysteinyl leukotrienes (cLTs) and eosinophil cationic protein (ECP) have been detected in airway secretions. ECP is a specific marker of eosinophil activation although leukotrienes can be released from a variety of cells including mast cells, eosinophils and monocytes. OBJECTIVE: To test the association between eosinophil activation and cysteinyl leukotriene production in the upper airway secretions of infants with RSV positive (RSV+ve) bronchiolitis. METHODS: Nasal lavage samples were performed in 78 infants (0.0-11.5 months) admitted to hospital with RSV+ve bronchiolitis soon after admission (0-48 h). Leukotriene C4 (LTC4) was assayed by enzyme immunoassay (EIA) and eosinophil cationic protein (ECP) by fluoroimmunoassay (FIA). RESULTS: LTC4 was detectable in 51 and ECP in 57 of 78 samples with a significant positive relationship between LTC4 and ECP (r=0.557, P<0.001). CONCLUSION: In the majority of our subjects with RSV+ve bronchiolitis ECP and LTC4 were detectable in upper airway secretions and were significantly associated with each other. In this clinical setting much of the detected LTC4 within upper airway secretions is likely to originate from the eosinophil, an observation that may have implications for clinical management and for delineation of the underlying mechanisms associated with this illness.

Importance of conserved acidic residues in mntH, the Nramp homolog of Escherichia coli.

A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters. Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E. coli Nramp homolog mntH. Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function. In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study. Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of (54)Mn(2+) uptake with a V(max) value of approximately 10% of wild-type and a slightly elevated K(m) value. Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn(2+). Other residues such as Glu-102 and Asp238 may play a role in the release of Mn(2+) to the cytoplasm or may be involved in maintaining secondary structure.

A face on transmembrane segment 8 of the lactose permease is important for transport activity.

Previous work on the lactose permease of Escherichia coli has shown that mutations along a face of predicted transmembrane segment 2 (TMS-2) play a critical role in conformational changes associated with lactose transport [Green, A. L., Anderson, E. J., and Brooker, R. J. (2000) J. Biol. Chem. 275, 23240-23246]. In the current study, mutagenesis was conducted along the side of predicted TMS-8 that contains the first amino acid in the conserved loop 8/9 motif. Several substitutions at positions 261, 265, 272, and 276 were markedly defective for downhill lactose transport although these mutants were well expressed. Substitutions along the entire side of TMS-8 containing the first amino acid in the loop 8/9 motif displayed defects in uphill lactose transport. Again, substitutions at positions 261, 265, 268, 272, and 276 were the most defective, with several of these mutants showing no lactose accumulation against a gradient. According to helical wheel plots, Phe-261, Thr-265, Gly-268, Asn-272, and Met-276 form a continuous stripe along one face of TMS-8. These results are discussed according to our hypothetical model, in which the two halves of the protein form a rotationally symmetrical dimer. In support of this model, alignment of predicted TMS-2 and TMS-8 shows an agreement between the amino acid residues in these transmembrane segments that are critical for lactose transport activities.

A triple mutant, K319N/H322Q/E325Q, of the lactose permease cotransports H+ with thiodigalactoside.

In a previous study, we characterized a lactose permease mutant (K319N/E325Q) that can transport H+ ions with sugar. This result was surprising because other studies had suggested that Glu-325 plays an essential role in H+ binding. To determine if the lactose permease contains one or more auxiliary H+ binding sites, we began with the K319N/E325Q strain, which catalyzes a sugar-dependent H+ leak, and isolated third site suppressor mutations that blocked the H+ leak. Three types of suppressors were obtained: H322Y, H322R, and M299I. These mutations blocked the H+ leak and elevated the apparent Km value for lactose. The M299I and H322Y suppressors could still transport H+ with beta-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H+/sugar cotransport. Four mutant strains containing a nonionizable substitution at codon 322 (H322Q) were analyzed. None of these were able to catalyze uphill accumulation of lactose, however, all showed some level of substrate-induced proton accumulation. The level seemed to vary based on the substrate being analyzed (lactose or TDG). Most interestingly, a triple mutant, K319N/H322Q/E325Q, catalyzed robust H+ transport with TDG. These novel results suggest an alternative mechanism of lactose permease cation binding and transport, possibly involving hydronium ion (H3O+).

The conserved motif in hydrophilic loop 2/3 and loop 8/9 of the lactose permease of Escherichia coli. Analysis of suppressor mutations.

The major facilitator superfamily (MFS) of transport proteins, which includes the lactose permease of Escherichia coli, contains a conserved motif G-X-X-X-D/E-R/K-X-G-R/K-R/K in the loops that connect transmembrane segments 2 and 3, and transmembrane segments 8 and 9. In three previous studies (Jessen-Marshall, A.E., & Brooker, R.J. 1996. J. Biol. Chem. 271:1400-1404; Jessen-Marshall, A.E., Parker, N., & Brooker, R.J. 1997. J. Bacteriol. 179:2616-2622; and Pazdernik, N., Cain, S.M., & Brooker, R.J. 1997. J. Biol. Chem. 272:26110-26116), suppressor mutations at twenty different sites were identified which restore function to mutant permeases that have deleterious mutations in the conserved loop 2/3 or loop 8/9 motif. In the current study, several of these second-site suppressor mutations have been separated from the original mutation in the conserved motif. The loop 2/3 suppressors were then coupled to a loop 8/9 mutation (P280L) and the loop 8/9 suppressors were coupled to a loop 2/3 mutation (i.e., G64S) to determine if the suppressors could restore function only to a loop 2/3 mutation, a loop 8/9 mutation, or both. The single parent mutations changing the first position in loop 2/3 (i.e., G64S) and loop 8/9 (i.e., P280L) had less than 4% lactose transport activity. Interestingly, most of the suppressors were very inhibitory when separated from the parent mutation. Two suppressors, A50T and G370V, restored substantial transport activity when individually coupled to the mutation in loop 2/3 and also when coupled to the corresponding mutation in loop 8/9. In other words, these suppressors could alleviate a defect imposed by mutations in either half of the permease. From a kinetic analysis, these suppressors were shown to exert their effects by increasing the V(max) values for lactose transport compared with the single G64S and P280L strains. These results are discussed within the context of our model in which the two halves of the lactose permease interact at a rotationally symmetrical interface, and that lactose transport is mediated by conformational changes at the interface.

A reaction-induced fourier transform-infrared spectroscopic study of the lactose permease. A transmembrane potential perturbs carboxylic acid residues.

In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions. This process occurs in all cells, but the microscopic mechanism is not understood. Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters. To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples. The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient. Positive bands in the 1765-1730 cm(-1) region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK(a), protonation state, or environment. This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level. Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.

A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes.

The lactose permease is an integral membrane protein that cotransports H(+) and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting the K(m) values (Jessen-Marshall, A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol. 179, 2616-2622). In the current study, mutagenesis was conducted along the face of TMS-2 that contains glycine-64. Single amino acid substitutions that substantially changed side-chain volume at codons 52, 57, 59, 63, and 66 had little or no effect on transport activity, whereas substitutions at codons 49, 53, 56, and 60 were markedly defective and/or had lower levels of expression. According to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64 form a continuous stripe along one face of TMS-2. Several of the TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as parental strains to isolate mutants that restore transport activity. These mutations were either first-site mutations or second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic analysis showed that the suppressors had a higher rate of lactose transport compared with the corresponding parental strains. Overall, the results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. We hypothesize that TMS-2 slides across TMS-7 and TMS-11 when the lactose permease interconverts between the C1 and C2 conformations. This idea is discussed within the context of a revised model for the structure of the lactose permease.

Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli.

The lactose permease is a polytopic membrane protein that has a duplicated conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), located in cytoplasmic loops 2/3 and 8/9. In the current study, the roles of the basic residues and the acidic residue were investigated in greater detail. Neutral substitutions of two positive charges in loop 2/3 were tolerated, while a triple mutant resulted in a complete loss of expression. Neutral substitutions of a basic residue in loop 8/9 (i.e., K289I) also diminished protein stability. By comparison, neutral substitutions affecting the negative charge in loop 2/3 had normal levels of expression, but were defective in transport. A double mutant (D68T/N284D), in which the aspartate of loop 2/3 was moved to loop 8/9, did not have appreciable activity, indicating that the negative charge in the conserved motif could not be placed in loop 8/9 to recover lactose transport activity. An analysis of site-directed mutants in loop 7/8 and loop 8/9 indicated that an alteration in the charge distribution across transmembrane segment 8 was not sufficient to alleviate a defect caused by the loss of a negative charge in loop 2/3. To further explore this phenomenon, the double mutant, D68T/N284D, was used as a parental strain to isolate suppressor mutations which restored function. One mutant was obtained in which an acidic residue in loop 11/12 was changed to a basic residue (i.e., Glu374 --> Lys). Overall, the results of this study suggest that the basic residues in the conserved motif play a role in protein insertion and/or stability, and that the negative charge plays a role in conformational changes.

A K319N/E325Q double mutant of the lactose permease cotransports H+ with lactose. Implications for a proposed mechanism of H+/lactose symport.

In this study, we have examined the transport characteristics of the wild-type lactose permease, single mutants in which Lys-319 was changed to asparagine or alanine or Glu-325 was changed to glutamine or alanine, and the corresponding double mutant strains. The wild-type and Asn-319 mutant showed high levels of lactose uptake, with Km values of 0.42 and 1.30 mM and Vmax values of 102.6 and 48.3 nmol of lactose/min/mg of protein, respectively. The Asn-319/Gln-325 strain had a normal Km of 0.36 mM and a moderate Vmax of 18.5 nmol of lactose/min/mg of protein. By comparison, the single E325Q strain had a normal Km of 0.27 mM but a very defective Vmax of 1.3 nmol of lactose/min/mg of protein. A similar trend was observed among the alanine substitutions at these positions, although the Vmax values were lower for the Ala-319 mutations. When comparing the Vmax values between the single position 325 mutants with those of the double mutants, these results indicate that neutral 319 mutations substantially alleviate a defect in Vmax caused by neutral 325 mutations. With regard to H+/lactose coupling, the wild-type permease is normally coupled and can transport lactose against a gradient. The position 325 single mutants showed no evidence of H+ transport with lactose or thiodigalactoside (TDG) and were unable to facilitate uphill lactose transport. The single Asn-319 mutant and double Asn-319/Gln-325 mutant were able to transport H+ upon the addition of lactose or TDG. In addition, both of these strains catalyzed a sugar-dependent H+ leak that inhibited cell growth in the presence of TDG. These two strains were also defective in uphill transport, which may be related to their sugar-dependent leak pathway. Based on these and other results in the literature, a model is presented that describes how the interactions among several ionizable residues within the lactose permease act in a concerted manner to control H+/lactose coupling. In this model, Lys-319 and Glu-325 play a central role in governing the ability of the lactose permease to couple the transport of H+ and lactose.

Fourier transform infrared analysis of purified lactose permease: a monodisperse lactose permease preparation is stably folded, alpha-helical, and highly accessible to deuterium exchange.

The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose symporter. In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques. The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag. In the novel purification method described here, lactose permease is purified from beta-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography. The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity. The protein has been successfully reconstituted and has been shown to be active for lactose transport. Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease. FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80% alpha-helical and stably folded at 20 degreesC; thermal denaturation is first detected at 70 degreesC. Because the purified protein is also readily susceptible to 2H exchange, these results suggest that the protein is conformationally flexible and that 2H exchange is facilitated as the result of conformational fluctuations from the folded state.

An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner.

A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N. J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild-type strain, but its Vmax for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the Vmax for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes.

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.

Role of conserved residues in hydrophilic loop 8-9 of the lactose permease.

A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane. Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. In a previous study concerning the loop 2-3 motif of the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995), it was shown that the first-position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport. In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif. In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an alpha-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered. In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop. With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated. Most substitutions produced permeases with normal or mildly defective transport activities. However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport. Kinetic analysis of position 280 mutants indicated that the defect decreased the Vmax for lactose uptake. Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates. The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.

Structural topology of transmembrane helix 10 in the lactose permease of Escherichia coli.

In the lactose permease of Escherichia coli, transmembrane helix 10 has been shown to be functionally important. The structure of this helix has been examined in greater detail in this study. A total of 46 substitution and 8 insertional mutants were constructed and analyzed along the entire length of transmembrane helix 10. The results identified amino acids that are tolerant of substitutions by a variety of amino acids. Since a number of these amino acids (Thr-320, Val-331, Phe-325, and Ile-317) are clustered in one region in a helical wheel projection of transmembrane helix 10, it seems likely that this face of helix 10 is interacting with the membrane. The channel lining domain is thought to consist of the helical face containing Glu-325, Leu-318, Leu-329, His-322, Val-315, Cys-333, Val-326, and Lys-319 based on the results here and from earlier findings. Deleterious mutations along this face tended to greatly increase the Km value for lactose transport with only minor effects on the Vmax. Analysis of insertional mutants revealed that perturbation of the spatial relationship between amino acids at the periplasmic edge is less deleterious than perturbation in the center of the helix or the cytoplasmic edge. Using all of the above information, a detailed structural topology of transmembrane helix 10 is proposed.

Evidence that transmembrane segment 2 of the lactose permease is part of a conformationally sensitive interface between the two halves of the protein.

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3. The motif is repeated again in the second half of the protein. In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A. E., Paul, N. J., and Brooker, R. J. (1995) J. Biol. Chem. 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport. In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function. A total of 10 different second site mutations were identified among a screen of 19 independent mutants. One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine. Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2. Surprisingly, the majority of suppressor mutations were found in the second half of the permease. All of these are expected to alter helix topology in either of two ways. Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234-->Trp or Phe, Gln-241-->Leu, and Phe-247-->Val) and TM-11 (Ser-366-->Phe). Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257-->Asp, Val-367-->Glu, Ala-369-->Pro, and a 5-codon insertion into loop 11/12). It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half. Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease. According to our tertiary model, this interaction occurs between TM-2 and TM-11.

The conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), in hydrophilic loop 2/3 of the lactose permease.

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine. Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high beta-turn propensity) may be structurally important at this position. The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity. In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low beta-turn propensity, still retained high levels of transport activity. Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues. However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose. Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this motif may be important in promoting global conformational changes within the permease.