Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.     

Characterization of the monomeric form of the transmembrane and cytoplasmic domains of the integrin beta 3 subunit by NMR spectroscopy

We have characterized a membrane protein containing residues P688-T762 of the integrin beta3 subunit, encompassing its transmembrane and cytoplasmic domains, by nuclear magnetic resonance spectroscopy. Under conditions in which it is monomeric in dodecylphosphocholine micelles, the protein consists mainly of alpha-helical structures. An amino-terminal helix corresponding to the beta3 transmembrane helix extends into the membrane-proximal region of the cytoplasmic domain. Moreover, following an apparent hinge at residues H722-D723, residues K725-A735 are mostly alpha-helical. In the presence of membrane-mimicking detergents, the cytoplasmic domain connected to the transmembrane helix is substantially ordered at pH 4.8 and 50 degrees C. Its carboxyl-terminal end takes on a turn-helix configuration characteristic of the immunoreceptor tyrosine-based activation motif. These structural features of the beta3 subunit should help to explain its interaction with numerous cytosolic interacting proteins and begin to illuminate the mechanism of integrin activation.     

Structure and dynamics of transmembrane signaling by the Escherichia coli aspartate receptor.

The structure of the cytosolic extension of the first transmembrane region (TM1) of the Escherichia coli aspartate receptor (residues 3, 4, and 5) and conformational changes within that region have been characterized by targeted cross-linking studies and by measurement of the effect of aspartate binding on cross-linking and methylation rates and compared with the periplasmic extension of the same helix. These experiments show that (1) the cytosolic extension of TM1 is helical, with residues 4 and 4' closest together at the dimer interface; (2) the helix is more solvent-exposed at the cytosolic side of the membrane than on the periplasmic side; and (3) aspartate binding enhances the rate of cross-linking at Cys 4, and the resulting cross-linked receptor displays aspartate-induced transmembrane increases in methylation by the cytoplasmic methylase (the CheR protein). We conclude that aspartate induces a conformational change that does not involve large intersubunit movements that lead to an increase in distance between the cytosolic ends of the first membrane-spanning helices; rather, the motion involved is largely contained within individual subunits, possibly resulting in a small movement between positions 4 and 4'.     

Subunit a facilitates aqueous access to a membrane-embedded region of subunit c in Escherichia coli F1F0 ATP synthase

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occurs in the middle of the membrane at Asp-61 on transmembrane helix 2 of subunit c. Previously, the reactivity of cysteines substituted into F(0) subunit a revealed two regions of aqueous access, one extending from the periplasm to the middle of the membrane and a second extending from the middle of the membrane to the cytoplasm. To further characterize aqueous accessibility at the subunit a-c interface, we have substituted Cys for residues on the cytoplasmic side of transmembrane helix 2 of subunit c and probed the accessibility to these substituted positions using thiolate-reactive reagents. The Cys substitutions tested were uniformly inhibited by Ag(+) treatment, which suggested widespread aqueous access to this generally hydrophobic region. Sensitivity to N-ethylmaleimide (NEM) and methanethiosulfonate reagents was localized to a membrane-embedded pocket surrounding Asp-61. The cG58C substitution was profoundly inhibited by all the reagents tested, including membrane impermeant methanethiosulfonate reagents. Further studies of the highly reactive cG58C substitution revealed that NEM modification of a single c subunit in the oligomeric c-ring was sufficient to cause complete inhibition. In addition, NEM modification of subunit c was dependent upon the presence of subunit a. The results described here provide further evidence for an aqueous-accessible region at the interface of subunits a and c extending from the middle of the membrane to the cytoplasm.     

F1F0-ATP synthase. Binding of delta subunit to a 22-residue peptide mimicking the N-terminal region of alpha subunit

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to alpha(3)beta(3) hexagon of F(1). Proteolysis and cross-linking had suggested that N-terminal residues of alpha subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of alpha ("alpha N1-22") binds specifically to isolated wild-type delta subunit with high affinity (K(d) = 130 nm), accounting for a major portion of the binding energy when delta-depleted F(1) and isolated delta bind together (K(d) = 1.4 nm). Stoichiometry of binding of alpha N1-22 to delta at saturation was 1/1, showing that in intact F(1)F(0)-ATP synthase only one of the three alpha subunits is involved in delta binding. When alpha N1-22 was incubated with delta subunits containing mutations in helices 1 or 5 on the F(1)-binding face of delta, peptide binding was impaired as was binding of delta-depleted F(1). Residues alpha 6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha 3-8. Circular dichroism measurements showed that alpha N1-22 had significant helical content. Hypothetically a helical region of residues alpha N1-22 packs with helices 1 and 5 on the F(1)-binding face of delta, forming the alpha/delta interface.     

The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism

Subunit a plays a key role in promoting H(+) transport and the coupled rotary motion of the subunit c ring in F(1)F(0)-ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(0) subunit c. H(+) are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Here we substituted Cys into loops connecting TMHs 1 and 2 (loop 1-2) and TMHs 3 and 4 (loop 3-4). A large segment of loop 3-4 extending from loop residue 192 loop to residue 203 in TMH4 at the lipid bilayer surface proved to be very sensitive to inhibition by Ag(+). Cys-161 and -165 at the other end of the loop bordering TMH3 were also sensitive to inhibition by Ag(+). Further Cys substitutions in residues 86 and 93 in the middle of the 1-2 loop proved to be Ag(+)-sensitive. We next asked whether the regions of Ag(+)-sensitive residues clustered together near the surface of the membrane by combining Cys substitutions from two domains and testing for cross-linking. Cys-161 and -165 in loop 3-4 were found to cross-link with Cys-202, -203, or -205, which extend into TMH4 from the cytoplasm. Further Cys at residues 86 and 93 in loop 1-2 were found to cross-link with Cys-195 in loop 3-4. We conclude that the Ag(+)-sensitive regions of loops 1-2 and 3-4 may pack in a single domain that packs at the ends of TMHs 3 and 4. We suggest that the Ag(+)-sensitive domain may be involved in gating H(+) release at the cytoplasmic side of the aqueous access channel extending through F(0).     

Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ

The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII. In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII. Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane. Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane.     

On the structure of the stator of the mitochondrial ATP synthase

The structure of most of the peripheral stalk, or stator, of the F-ATPase from bovine mitochondria, determined at 2.8 A resolution, contains residues 79-183, 3-123 and 5-70 of subunits b, d and F6, respectively. It consists of a continuous curved alpha-helix about 160 A long in the single b-subunit, augmented by the predominantly alpha-helical d- and F6-subunits. The structure occupies most of the peripheral stalk in a low-resolution structure of the F-ATPase. The long helix in subunit b extends from near to the top of the F1 domain to the surface of the membrane domain, and it probably continues unbroken across the membrane. Its uppermost region interacts with the oligomycin sensitivity conferral protein, bound to the N-terminal region of one alpha-subunit in the F1 domain. Various features suggest that the peripheral stalk is probably rigid rather than resembling a flexible rope. It remains unclear whether the transient storage of energy required by the rotary mechanism takes place in the central stalk or in the peripheral stalk or in both domains.     

Transmembrane organization of the Bacillus subtilis chemoreceptor McpB deduced by cysteine disulfide crosslinking

The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.     

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.     

Light-induced structural changes occur in the transmembrane helices of the Natronobacterium pharaonis HtrII transducer.

The Natronobacterium pharaonis HtrII (NpHtrII) transducer interacts with its cognate photoactive sensory rhodopsin receptor, NpSRII, to mediate phototaxis responses. NpHtrII is predicted to have two transmembrane helices and a large cytoplasmic domain and to form a homodimer. Single cysteines were substituted into an engineered cysteine-less NpHtrII at 38 positions in its transmembrane domain. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of NpSRII. The rapid cross-linking rates at several positions support that NpHtrII is a dimer when functionally expressed in the Halobacterium salinarum membrane. Thirteen positions in the second transmembrane segment (TM2) exhibited significant light-induced increases in cross-linking efficiency, and they define a single face traversing the length of the segment when modeled as an alpha-helix. Four positions in this helix showing light-induced decreases in efficiency are clustered on the cytoplasmic side of the protein. One of the monocysteine mutants, G83C, showed loss of phototaxis responses, and analysis of double mutants showed that the G83C mutation alters the dark structure of the TM2-TM2' region of NpHtrII. In summary, the results reveal conformationally active regions in the second transmembrane segment of NpHtrII and a face along the length of TM2 that becomes more available for TM2-TM2' cross-linking upon receptor photoactivation. The data also establish that one residue in TM2, Gly83, is critical for maintaining the proper conformation of NpHtrII for signal relay from the photoactivated receptor to the kinase-binding region of the transducer.     

Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase

The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.     

The mitochondrial citrate transport protein: probing the secondary structure of transmembrane domain III, identification of residues that likely comprise a portion of the citrate transport pathway, and development of a model for the putative TMDIII-TMDIII' interface

The mitochondrial citrate transport protein (CTP) has been investigated by mutating 28 consecutive residues within transmembrane domain III (TMDIII), one at a time, to cysteine. A cysteine-less CTP that retains wild-type functional properties, served as the starting template. The single Cys CTP mutants were abundantly expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to two methanethiosulfonate reagents was evaluated by determining the rate constants for inhibition of CTP function. These rate constants varied by over five orders of magnitude. With two independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of 4 was observed from residues 123-137. Based on the pattern of accessibility we conclude that residues 123-137 exist as an alpha-helix. Although less certain, a combination of the rate constant data and the specific activity data with the single Cys mutants suggests that the alpha-helical secondary structure may extend to residue 113. Furthermore, the rate constant data define water-accessible and water-inaccessible faces of the helix. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer. Finally, based on a combination of the CTP inhibition rate constant data and the existence of significant sequence identity with a transmembrane segment within glycophorin A that forms a portion of its dimer interface, a model for a putative CTP TMDIII-TMDIII' dimer interface has been developed.     

Structural characterization of the transmembrane domain from subunit e of yeast F1Fo-ATP synthase: a helical GXXXG motif located just under the micelle surface

F1Fo-ATP synthase is a large multiprotein complex, including at least 10 subunits in the membrane-bound Fo-sector. One of these Fo proteins is subunit e (Su e), involved in the stable dimerization of F1Fo-ATP synthase, and required for the establishment of normal cristae membrane architecture. As a step toward enabling structure-function studies of the Fo-sector, the Su e transmembrane region was structurally characterized in micelles. Based on a series of NMR and CD (circular dichroism) studies, a structural model of the Su e/micelle complex was constructed, indicating Su e is largely helical, and emerges from the micelle with Arg20 near the phosphate head groups. Su e only adopts this folded conformation in the context of the micelle, and is essentially disordered in DMSO, water or trifluoroethanol/water. Within the micelle the C-terminal Ala10-Arg20 stretch is helical, while the region N-terminal may be transiently helical, based on negative CSI (chemical shift index) values. The Ala10-Arg20 helix contains the G14XXXG18 motif, which has been proposed to play an important role in dimer formation with another protein from the Fo-sector. The Gly on the C-terminal end of this motif (Gly18) is slightly more mobile than the more buried Gly14, based on NMR order parameter measurements (Gly14 S2 = 0.950; Gly18 S2 = 0.895). Only one Su e transmembrane peptide is bound per micelle, and micelles are 22-23 A in diameter, composed of 51 +/- 4 dodecylphosphocholine detergent molecules. Although there is no evidence for Su e homodimerization via the transmembrane domain, potentially synergistic roles for N-terminal (membrane) and C-terminal (soluble) domain interactions may still occur. Furthermore, the presence of a buried charged residue (Arg7) suggests there may be interactions with other Fo-sector protein(s) that stabilize this charge, and possibly drive the folding of the N-terminal 9 residues of the transmembrane domain.     

Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme

The transmembrane sector of the F(0)F(1) rotary ATP synthase is proposed to organize with an oligomeric ring of c subunits, which function as a rotor, interacting with two b subunits at the periphery of the ring, the b subunits functioning as a stator. In this study, cysteines were introduced into the C-terminal region of subunit c and the N-terminal region of subunit b. Cys of N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of subunit c. In each case, a maximum of 50% of the b subunit could be cross-linked to subunit c, which suggests that either only one of the two b subunits lie adjacent to the c-ring or that both b subunits interact with a single subunit c. The results support a topological arrangement of these subunits, in which the respective N- and C-terminal ends of subunits b and c extend to the periplasmic surface of the membrane and cAsp-61 lies at the center of the membrane. The cross-linking of Cys between bN2C and cV78C was shown to inhibit ATP-driven proton pumping, as would be predicted from a rotary model for ATP synthase function, but unexpectedly, cross-linking did not lead to inhibition of ATPase activity. ATP hydrolysis and proton pumping are therefore uncoupled in the cross-linked enzyme. The c subunit lying adjacent to subunit b was shown to be mobile and to exchange with c subunits that initially occupied non-neighboring positions. The movement or exchange of subunits at the position adjacent to subunit b was blocked by dicyclohexylcarbodiimide. These experiments provide a biochemical verification that the oligomeric c-ring can move with respect to the b-stator and provide further support for a rotary catalytic mechanism in the ATP synthase.     

Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli

Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.     

Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit

The stator in F(1)F(o)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(o) and to the alpha(3)beta(3) hexagon of F(1). Previous work has shown that N-terminal residues of alpha subunit are involved in binding delta. A synthetic peptide consisting of the first 22 residues of alpha (alphaN1-22) binds specifically to isolated wild-type delta subunit with 1:1 stoichiometry and high affinity, accounting for a major portion of the binding energy between delta and F(1). Residues alpha6-18 are predicted by secondary structure algorithms and helical wheels to be alpha-helical and amphipathic, and a potential helix capping box occurs at residues alpha3-8. We introduced truncations, deletions, and mutations into alphaN1-22 peptide and examined their effects on binding to the delta subunit. The deletions and mutations were introduced also into the N-terminal region of the uncA (alpha subunit) gene to determine effects on cell growth in vivo and membrane ATP synthase activity in vitro. Effects seen in the peptides were well correlated with those seen in the uncA gene. The results show that, with the possible exception of residues close to the initial Met, all of the alphaN1-22 sequence is required for binding of delta to alpha. Within this sequence, an amphipathic helix seems important. Hydrophobic residues on the predicted nonpolar surface are important for delta binding, namely alphaIle-8, alphaLeu-11, alphaIle-12, alphaIle-16, and alphaPhe-19. Several or all of these residues probably make direct interaction with helices 1 and 5 of delta. The potential capping box sequence per se appeared less important. Impairment of alpha/delta binding brings about functional impairment due to reduced level of assembly of ATP synthase in cells.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.     

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.     

The polar domain of the b subunit of Escherichia coli F1F0-ATPase forms an elongated dimer that interacts with the F1 sector.

A soluble form of the b subunit of the F0 sector of the F1F0-ATPase of Escherichia coli has been produced, purified, and characterized. In this form of the protein, designated bsol, residues 25-146 (the carboxyl terminus) of b have been fused to an amino-terminal octapeptide extension derived from the vector pUC8. The inferred subunit molecular weight of bsol is 15,459. bsol protein was expressed in E. coli as a soluble cytoplasmic protein and was readily purified to homogeneity by conventional methods. The molecular weight of bsol, determined by sedimentation equilibrium, was 31,200, indicating that the protein is dimeric. Chemical cross-linking studies supported this conclusion. However, bsol sedimented with a coefficient of just 1.8 S and behaved on size exclusion chromatography with an apparent molecular weight of 80,000-85,000. These results indicate that the protein exists in solution as a highly elongated dimer. The circular dichroism spectrum indicated that bsol is highly alpha-helical. Binding of bsol to F1-ATPase was directly demonstrated by size exclusion chromatography. bsol also inhibited the binding of F1-ATPase to F1-depleted membrane vesicles, as measured by reconstitution of energy-dependent quinacrine fluorescence quenching. This result implies that bsol and F0 compete for binding to the same site on F1. The apparently normal interaction of bsol with F1-ATPase strongly suggests that the recombinant protein assumes the correct structure. No substantial effects of bsol on the ATPase activity of purified F1 were observed.