Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. off

Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.     

Cysteine-scanning mutagenesis around transmembrane segment III of Tn10-encoded metal-tetracycline/H+ antiporter

Each amino acid in the putative transmembrane helix III and its flanking regions (from Gly-62 to Tyr-98) of the Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) was individually replaced with Cys. Out of these 37 cysteine-scanning mutants, the mutants from G62C to R70C and from S92C to Y98C showed high or intermediate reactivity with [14C]N-ethylmaleimide (NEM) except for the M64C mutant. On the other hand, the mutants from R71C to S91C showed almost no reactivity with NEM except for the P72C mutant. These results confirm that the transmembrane helix III is composed of 21 residues from Arg-71 to Ser-91. The majority of Cys replacement mutants retained high or moderate tetracycline transport activity. Cys replacements for Gly-62, Asp-66, Ser-77, Gly-80, and Asp-84 resulted in almost inactive Tet(B) (less than 3% of the wild-type activity). The Arg-70 --> Cys mutant retained very low activity due to a mercaptide between Co2+ and a SH group (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Three of these six important residues (Ser-77, Gly-80, and Asp-84) are located in the transmembrane helix III and one (Arg-70) is located in the flanking region. These four functionally important residues are located on one side of the helical wheel. Only two of the residual 31 Cys mutants were inactivated by NEM (S65C and L97C). Ser-65 and Leu-97 are located on the cytoplasmic and periplasmic loops, respectively, in the topology of Tet(B). The degree of inactivation of these Cys mutants with SH reagents was dependent on the volume of substituents. In the presence of tetracycline, the reactivity of the S65C mutant with NEM was significantly increased, in contrast, the reactivity of L97C was greatly reduced, indicating that the cytoplasmic and periplasmic loop regions undergo substrate-induced conformational change in the mutually opposite direction.     

The yeast mic2 mutant is defective in the formation of mannosyl-diinositolphosphorylceramide

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243-246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp74, Asp200, Asp318 and Glu381 are located on the putative cytoplasmic side, and Asp39 and Glu345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu345 and Glu381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp318 is located in cytoplasmic loop 10-11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2-3 [Yamaguchi et al., Biochemistry 31 (1992) 8344-8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.     

Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.     

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.     

The substrate-binding site in the lactose permease of Escherichia coli

Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 --> Cys was also studied. In marked contrast to Val-331 --> Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 --> Ala/Val-331 --> Cys, Arg-144 --> Ala/Val-331 --> Cys, or Arg-144 --> Lys/Val-331 --> Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 --> Ala or Arg-144 --> Ala and, to a lesser extent, Arg-144 --> Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I-. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H+ translocation pathway at the interface between helices IX and X.     

Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutant merT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Gly14Arg, Gly15Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity

Chemical modification studies and site-directed mutagenesis experiments have provided evidence that human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) are serine esterases. Loss of HLAL and HGL activity was also observed in the presence of sulfhydryl-reactive substances, suggesting that cysteines are likewise essential for substrate hydrolysis. To study the functional role of the HLAL and HGL cysteine residues, we replaced these amino acids with alanine by site-directed mutagenesis. Substitutions at positions 227 and 236, alone or together, drastically reduced hydrolytic activity in a substrate-dependent manner while the other mutants were not affected to any great extent. HLAL(Cys227-->Ala), HLAL(Cys236-->Ala), and HLAL(Cys227-->Ala, Cys236-->Ala) were essentially inactive against cholesteryl oleate, but retained about 23-39%, 28-37%, and 13-17% of catalytic activity for both triolein and tributyrin, respectively. The data obtained with the corresponding HGL mutants confirmed the importance of residues 227 and 236 in maintaining enzymatic activity towards long- and short-chain triglycerides. In order to assess the contribution of the eight amino acids delimited by Cys227 and Cys236 to lipolysis, we generated HLAL replacement mutants containing the corresponding residues 228-235 of HGL or RLL. Both HLAL chimeras were catalytically active towards all three substrates analyzed, indicating that these amino acids do not determine HLAL substrate specificity. Deletion of the eight-amino acid alpha-helix as well as disruption of its hydrophobic surface, in contrast, abolished enzymatic activity. Our studies suggest that Cys227, Cys236, and the amphipathic helix formed by residues 228-235 are essential for HLAL- and HGL-mediated neutral lipid catabolism.     

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.     

Functional mapping of the surface residues of human thrombin

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.     

Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli

By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles. In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment. In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect to the N-terminal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while ligand binding causes much greater increases in reactivity. The findings indicate that 4B1 binding induces a structural change in the permease that is much less widespread than that induced by ligand binding.     

Cysteine scanning mutagenesis of helix V in the lactose permease of Escherichia coli

Using a functional lactose permease mutant devoid of Cys (C-less permease), each amino acid residue in putative transmembrane helix V was replaced individually with Cys (from Met145 to Thr163). Of the 19 mutants, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-less permease). Cys replacement of Gly147 or Trp151 essentially inactivates the permease (< 10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030] demonstrate that neither of these residues is important for activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to C-less permease with the exception of Trp151-->Cys and single Cys154 permeases which are present in reduced amounts. Finally, only three of the single-Cys mutants are inactivated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.     

Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli

Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273-->Cys/Met299-->Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.     

Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization

The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Delta8-Delta7 isomerization-deficient erg2-3 yeast strain. Ala scanning mutagenesis was used to identify residues in the four putative transmembrane alpha-helices of hEBP that are required for catalytic activity. Isomerization was assayed in vivo by spectrophotometric quantification of Delta5,7-sterols. Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A-, and W197A-expressing yeast strains contained 10% or less of wild-type (wt) Delta5,7-sterols. All substitutions of these six residues with functionally or structurally similar amino acid residues failed to fully restore catalytic activity. Mutants E81D, T126S, N194Q, and W197F, but not H77N and E123D, still bound the enzyme inhibitor 3H-ifenprodil. Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding [Moebius et al. (1996) Biochemistry 35, 16871-16878]. His77, Glu81, Glu123, Thr126, Asn194, and Trp197 are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are proposed to line the catalytic cleft. Ala mutants of Trp102, Tyr105, Asp109, Arg111, and Tyr112 in a conserved cytoplasmic domain (WKEYXKGDSRY) between transmembrane segments 2 and 3 contained less than 10% of wt Delta5,7-sterols, implying that this region also could be functionally important. The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional consequences of mutations in sterol modifying enzymes.     

The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling

The ligand binding site of neuropeptide Y (NPY) at the rat Y1 (rY1,) receptor was investigated by construction of mutant receptors and [3H]NPY binding studies. Expression levels of mutant receptors that did not bind [3H]NPY were examined by an immunological method. The single mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abolished [3H]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [3H]NPY binding. Similarly, the double mutation Leu34Arg/Asp199Ala totally abrogated the binding of [3H]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [3H]NPY 2.7- and 5.2-fold, respectively. The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affected [3H]NPY binding. A receptor with a deletion of the segment Asn2-Glu20 or with simultaneous mutations of the three putative N-terminal glycosylation sites, displayed no detectable [3H]NPY binding, due to abolished expression of the receptor at the cell surface. Taken together, these results suggest that amino acids in the N-terminal part as well as in the first and second extracellular loops are important for binding of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a proper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial for correct expression of the rY1 receptor at the cell surface.     

Site-directed chemical modification of cysteine-scanning mutants as to transmembrane segment II and its flanking regions of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a transmembrane water-filled channel

Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H+ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II. None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation. [14C]N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated. The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A. (1997) J. Biol. Chem. 272, 580-585). However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with [14C]NEM. The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment. Positions reactive to NEM appear periodically in the second half. They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel. The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half. These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway.     

Structural basis for molecular recognition between nuclear transport factor 2 (NTF2) and the GDP-bound form of the Ras-family GTPase Ran

Nuclear transport factor 2 (NTF2) and the Ras-family GTPase Ran are two soluble components of the nuclear protein import machinery. NTF2 binds GDP-Ran selectively and this interaction is important for efficient nuclear protein import in vivo. We have used X-ray crystallography to determine the structure of the macromolecular complex formed between GDP-Ran and nuclear transport factor 2 (NTF2) at 2.5 A resolution. The interaction interface involves primarily the putative switch II loop of Ran (residues 65 to 78) and the hydrophobic cavity and surrounding surface of NTF2. The major contribution to the interaction made by the switch II loop accounts for the ability of NTF2 to discriminate between GDP and GTP-bound forms of Ran. The aromatic side-chain of Ran Phe72 inserts into the NTF2 cavity and accounts for 22% of the surface area buried by the interaction interface, while salt bridges are formed between Lys71 and Arg76 of Ran with Asp92/Asp94 and Glu42 of NTF2, respectively. These salt bridges account for the inhibition of the Ran-NTF2 interaction by NTF2 mutants such as E42 K and D92/94N in which the negatively charged residues surrounding the cavity were altered. Because the interaction interface maintains the positions of key Ran residues involved in binding MgGDP, NTF2 binding may help stabilize the switch state of Ran, possibly in the context of targeting it to other components of the nuclear protein import machinery to specify directionality of transport. The binding of GDP-Ran at the NTF2 cavity raises the possibility that this interaction might be modulated by a metabolite or small molecule substrate for NTF2's putative enzymatic activity.     

Identification of a phosphate regulatory site and a low affinity binding site for glucose 6-phosphate in the N-terminal half of human brain hexokinase

Crystal structures of human hexokinase I reveal identical binding sites for phosphate and the 6-phosphoryl group of glucose 6-phosphate in proximity to Gly87, Ser88, Thr232, and Ser415, a binding site for the pyranose moiety of glucose 6-phosphate in proximity to Asp84, Asp413, and Ser449, and a single salt link involving Arg801 between the N- and C-terminal halves. Purified wild-type and mutant enzymes (Asp84 --> Ala, Gly87 --> Tyr, Ser88 --> Ala, Thr232 --> Ala, Asp413 --> Ala, Ser415 --> Ala, Ser449 --> Ala, and Arg801 --> Ala) were studied by kinetics and circular dichroism spectroscopy. All eight mutant hexokinases have kcat and Km values for substrates similar to those of wild-type hexokinase I. Inhibition of wild-type enzyme by 1,5-anhydroglucitol 6-phosphate is consistent with a high affinity binding site (Ki = 50 microM) and a second, low affinity binding site (Kii = 0.7 mM). The mutations of Asp84, Gly87, and Thr232 listed above eliminate inhibition because of the low affinity site, but none of the eight mutations influence Ki of the high affinity site. Relief of 1,5-anhydroglucitol 6-phosphate inhibition by phosphate for Asp84 --> Ala, Ser88 --> Ala, Ser415 --> Ala, Ser449 --> Ala and Arg801 --> Ala mutant enzymes is substantially less than that of wild-type hexokinase and completely absent in the Gly87 --> Tyr and Thr232 --> Ala mutants. The results support several conclusions. (i) The phosphate regulatory site is at the N-terminal domain as identified in crystal structures. (ii) The glucose 6-phosphate binding site at the N-terminal domain is a low affinity site and not the high affinity site associated with potent product inhibition. (iii) Arg801 participates in the regulatory mechanism of hexokinase I.     

Positively and negatively charged residues have different effects on the position in the membrane of a model transmembrane helix

We have studied the effects of single charged residues on the position of a model transmembrane helix in the endoplasmic reticulum membrane using the glycosylation mapping technique. Asp and Glu residues cause a re-positioning of the C-terminal end of the transmembrane helix when placed in the one to two C-terminal turns but not when placed more centrally. Arg and Lys residues, in contrast, have little effect when placed in the two C-terminal turn but give rise to a more substantial shift in position when placed 9-11 residues from the helix end. We suggest that this difference between the effects of positively and negatively charged residues can be explained by the so-called snorkel effect, i.e. that the very long side-chains of Arg and Lys can reach up along the transmembrane helix to allow the terminal, charged moiety to reside in the lipid headgroup region while the Calpha of the residue is positioned well below the membrane/water interface.