Structure-based functional study reveals multiple roles of transmembrane segment IX and loop VIII-IX in NhaA Na+/H+ antiporter of Escherichia coli at physiological pH

The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na(+)/H(+) antiporter. However, because NhaA is activated at physiological pH (pH 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu(241)-Phe(267)) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H(+)/Na(+) stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na(+), suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu(255) and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10-18 A) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-A cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K(m) for Na(+) of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.     

Helix packing in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking: helix I is close to helices V and XI

Coexpression of lacY gene fragments encoding the first two transmembrane domains and the remaining 10 transmembrane domains complement in the membrane and catalyze active lactose transport [Wrubel, W., Stochaj, U., et al. (1990) J. Bacteriol. 172, 5374-5381]. Accordingly, a plasmid encoding contiguous, nonoverlapping permease fragments with a discontinuity in the cytoplasmic loop between helices II and III (loop II/III) was constructed (N2C10 permease). When Phe27 (helix I) is replaced with Cys, cross-linking is observed with two native Cys residues, Cys148 (helix V) and Cys355 (helix XI). Cross-linking of a Cys residue at position 27 to Cys148 occurs with N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), with N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A), or with 1,6-bis(maleimido)hexane (BMH; flexible 16 A). On the other hand, with the Phe27-->Cys/Cys355 pair, cross-linking is observed with p-PDM or BMH but not o-PDM. In neither case is cross-linking observed with iodine. It is suggested that a Cys residue at position 27 is within 6-10 A from Cys148 and about 10 A from Cys355. The results provide evidence for proximity between helix I and helices V or XI in the tertiary structure of the permease. In addition, the findings are consistent with other results [Venkatesan, P., Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] indicating that Glu126 (helix IV) and Arg144 (helix V) are within the membrane, rather than at the membrane-water interface on the cytoplasmic face.     

Unraveling functional and structural interactions between transmembrane domains IV and XI of NhaA Na+/H+ antiporter of Escherichia coli

A functionally important, interface domain between transmembrane segments (TMSs) IV and XI of the NhaA Na+/H+ antiporter of Escherichia coli has been unraveled. Scanning by single Cys replacements identified new mutations (F136C, G125C, and A137C) that cluster in one face of TMS IV and increase dramatically the Km of the antiporter. Whereas G125C, in addition, causes a drastic alkaline shift to the pH dependence of the antiporter, G338C alleviates the pH control of NhaA. Scanning by double Cys replacements (21 pairs of one replacement per TMS) identified genetically eight pairs of residues that showed very strong negative complementation. Cross-linking of the double mutants identified six double mutants (T132C/G338C, D133C/G338C, F136C/S342C, T132C/S342C, A137C/S342C, and A137C/G338C) of which pronounced intramolecular cross-linking defined an interface domain between the two TMSs. Remarkably, cross-linking by a short and rigid reagent (N,N'-o-phenylenedimaleimide) revived the Li+/H+ antiport activity, whereas a shorter reagent (1,2-ethanediyl bismethanethiosulfonate) revived both Na+/H+ and Li+/H+ antiporter activities and even the pH response of the dead mutant T132C/G338C. Hence, cross-linking at this position restores an active conformation of NhaA.     

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na⁺/H⁺ antiporter. However, because NhaA is activated at physiological pH (pH 5.5–8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K_m to Na⁺ and Li⁺ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [¹⁴C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent K_m of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp⁶⁵ negative charge for pH activation of the antiporter.     

Functional and structural interactions of the transmembrane domain X of NhaA, Na+/H+ antiporter of Escherichia coli, at physiological pH

The 3D structure of Escherichia coli NhaA, determined at pH 4, provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 7.0-8.5), many questions pertaining to the active state of NhaA have remained open, including the physiological role of helix X. Using a structural-based evolutionary approach in silico, we identified a segment of most conserved residues in the middle of helix X. These residues were then used as targets for functional studies at physiological pH. Cysteine-scanning mutagenesis showed that Gly303, in the middle of the conserved segment, is an essential residue and Cys replacement of Lys300 retains only Li+/H+ antiporter activity, with a 20-fold increase in the apparent KM for Li+. Cys replacements of Leu296 and Gly299 increase the apparent KM of the Na+/H+ antiporter for both Na+ and Li+. Accessibility test to N-ethylmaleimide and 2-sulfonatoethyl methanethiosulfonate showed that G299C, K300C, and G303C are accessible to the cytoplasm. Suppressor mutations and site-directed chemical cross-linking identified a functional and/or structural interaction between helix X (G295C) and helix IVp (A130C). While these results were in accordance with the acid-locked crystal structure, surprisingly, conflicting data were also obtained; E78C of helix II cross-links very efficiently with several Cys replacements of helix X, and E78K/K300E is a suppressor mutation of K300E. These results reveal that, at alkaline pH, the distance between the conserved center of helix X and E78 of helix II is drastically decreased, implying a pH-induced conformational change of one or both helices.     

Location of helix III in the lactose permease of Escherichia coli as determined by site-directed thiol cross-linking

The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227, 228, 231, 232, 235, 238, 239, 241, 243, 245, or 246) was examined by using iodine or two rigid homobifunctional thiol-specific cross-linking reagents with different lengths [N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N, N'-p-phenylenedimaleimide (p-PDM; 10 A)]. Cys residues in the periplasmic half of helix III (position 87, 93, or 96) cross-link to Cys residues in the periplasmic half of helix VII (position 235, 238, 239, 241, or 245). In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices III (position 78 or 81) and VII (position 227, 228, 213, 232, or 235). Therefore, the periplasmic halves of helices III and VII are in close proximity, and the helices tilt away from each other toward the cytoplasmic face of the membrane. On the basis of the findings, a modified helix packing model for the permease is presented.     

The proximity of the C-terminal domain of Escherichia coli ribosomal protein L7/L12 to L10 determined by cysteine site-directed mutagenesis and protein-protein cross-linking.

Oligonucleotide-directed mutagenesis was used to produce a serine 89 to cysteine 89 substitution in the C-terminal globular domain of Escherichia coli ribosomal protein L7/L12. Cys-89 represented the only cysteine residue in the protein. L7/L12Cys89 was overproduced in E. coli and purified. An allele replacement strain was also constructed. Growth of this strain was indistinguishable from that of wild type. Ribosomes from the allele replacement strain were used to determine the location of the C-terminal domains of L7/L12 by disulfide cross-linking. A new homobifunctional cysteine-specific cross-linking reagent, 1,4-di[3'-(2'-pyridyldithio)-propionamido]butane, and diagonal gel electrophoresis were used to identify ribosomal proteins cross-linked to L7/L12Cys89. A cross-link between L7/L12 and the single cysteine in L10 was found, in addition to L7/L12 dimers. The L7/L12Cys89-L10 cross-link locates the C-terminal domain of at least one L7/L12 dimer on the body of the large subunit and supports our previous model (Olson, H. M., Sommer, A., Tewari, D. S., Traut, R. R., and Glitz, D. G. (1986) J. Biol. Chem. 261, 6924-6932) that depicts one of the two dimers of L7/L12 on the surface of the body of the 50 S subunit in a bent conformation with the C-terminal domain in close proximity to the N-terminal domain at the base of the stalk.     

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.     

Rhodopsin forms a dimer with cytoplasmic helix 8 contacts in native membranes

G protein-coupled receptors form dimers and higher-order oligomers in membranes, but the precise mode of receptor-receptor interaction remains unknown. To probe the intradimeric proximity of helix 8 (H8), we conducted chemical cross-linking of endogenous cysteines in rhodopsin in disk membranes. We identified a Cys316-Cys316 cross-link using partial proteolysis and liquid chromatography with mass spectrometry. These results show that a symmetric dimer interface mediated by H1 and H8 contacts is present in native membranes.     

Mutation E252C increases drastically the Km value for Na+ and causes an alkaline shift of the pH dependence of NhaA Na+/H+ antiporter of Escherichia coli

A single Cys replacement of Glu at position 252 (E252C) in loop VIII-IX of NhaA increases drastically the Km for Na(+) (50-fold) of the Na(+)/H(+) antiporter activity of NhaA and shifts the pH dependence of NhaA activity, by one pH unit, to the alkaline range. In parallel, E252C causes a similar alkaline pH shift to the pH-induced conformational change of loop VIII-IX. Thus, although both the Na(+)/H(+) antiporter activity of wild type NhaA and its accessibility to trypsin at position Lys(249) in loop VIII-IX increase with pH between pH 6.5 and 7.5, the response of E252C occurs above pH 8. Furthermore, probing accessibility of pure E252C protein in dodecyl maltoside solution to 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid revealed that E252C itself undergoes a pH-dependent conformational change, similar to position Lys(249), and the rate of the pH-induced conformational change is increased specifically by the presence of Na(+) or Li(+), the specific ligands of the antiporter. Chemical modification of E252C by N-ethylmaleimide, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid; [2-(trimethylammonium)ethyl]methane thiosulfonate, or (2-sulfonatoethyl)methanethiosulfonate reversed, to a great extent, the pH shift conferred by E252C but had no effect on the K(m) of the mutant antiporter.     

Site-directed chemical cross-linking demonstrates that helix IV is close to helices VII and XI in the lactose permease

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied. Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A). Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243). In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246). On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link. The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI.     

A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside.

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column. Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.     

Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences

Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli has been obtained [Williams, K. A., Kaufer, U. G., Padan, E., Schuldiner, S. and Kühlbrandt, W. (1999) EMBO J., 18, 3558-3563]. In these crystals NhaA exists as a dimer. Using biochemical and genetic approaches here we show that NhaA exists in the native membrane as a homooligomer. Functional complementation between the polypeptides of NhaA was demonstrated by coexpression of pairs of conditional lethal (at high pH in the presence of Na+) mutant alleles of nhaA in EP432, a strain lacking antiporters. Physical interaction in the membrane was shown between the His-tagged NhaA polypeptide which is readily affinity purified from DM-solubilized membranes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column. The organization of the oligomer in the membrane was studied in situ by site-directed cross-linking experiments. Cysteine residues were introduced--one per NhaA--into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place. Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE. The results are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers. Intermolecular cross-linking of V254C caused an acidic shift in the pH profile of NhaA.     

Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli

Membrane transporter ProP from Escherichia coli senses extracellular osmolality and responds by mediating the uptake of osmoprotectants such as glycine betaine when osmolality is high. Earlier EPR and NMR studies showed that a peptide replica of the cytoplasmic ProP carboxyl terminus (residues D468-R497) forms a homodimeric, antiparallel, alpha-helical coiled coil in vitro stabilized by electrostatic interactions involving R488. Amino acid replacement R488I disrupted coiled-coil formation by the ProP peptide, elevated the osmolality at which ProP became active, and rendered the osmolality response of ProP transient. In the present study, either E480 or K473 was replaced with cysteine (Cys) in ProP, a Cys-less, fully functional, histidine-tagged ProP variant, to use Cys-specific cross-linking approaches to determine if antiparallel coiled-coil formation and dimerization of the intact protein occur in vivo. The Cys at positions 480 would be closer in an antiparallel dimer than those at positions 473. These replacements did not disrupt coiled-coil formation by the ProP peptide. Partial homodimerization of variant ProP-E480C could be demonstrated in vivo and in membrane preparations via Cys-specific cross-linking with dithiobis(maleimidoethane) or by Cys oxidation to cystine by copper phenanthroline. In contrast, these reagents did not cross-link ProP with Cys at position 133 or 241. Cross-linking of ProP with Cys at position 473 was limited and occurred only if ProP was overexpressed, consistent with an antiparallel orientation of the coiled coil in the intact protein in vivo. Although replacement E480C did not alter transporter activity, replacement K473C reduced the extent and elevated the threshold for osmotic activation. K473 may play a role in ProP structure and function that is not reflected in altered coiled-coil formation by the corresponding peptide. Substitution R488I affected the activities of ProP-(His)(6), ProP-E480C, and ProP-K473C as it affected the activity of ProP. Surprisingly, it did not eliminate cross-linking of Cys at position 480, and it elevated cross-linking at position 473, even when ProP was expressed at physiological levels. This suggested that the R488I substitution may have changed the relative orientation of the C-termini within the dimeric protein from antiparallel to parallel, resulting in only transient osmotic activation. These results suggest that ProP is in monomer-dimer equilibrium in vivo. Dimerization may be mediated by C-terminal coiled-coil formation and/or by interactions between other structural domains, which in turn facilitate C-terminal coiled-coil formation. Antiparallel coiled-coil formation is required for activation of ProP at low osmolality.     

Proximity relationships between helices I and XI or XII in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking.

The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied. Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used. Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three homobifunctional thiol-specific cross-linking reagents of different lengths and flexibilities (6 A, rigid; 10 A, rigid; 16 A, flexible) or iodine. Cys residues in the periplasmic half of helix I cross-link to Cys residues in the periplasmic half of helix XI. In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices I and XI. Therefore, the periplasmic halves of helices I and XI are in close proximity, and the helices tilt away from each other towards the cytoplasmic face of the membrane. Cross-linking is also found with paired Cys residues near the middle of helices I and XII, but not with paired Cys residues near either end of the helices. Thus, helices I and XII are in close proximity only in the approximate middle of the membrane. Based on the findings, a modified helix packing model is proposed.     

Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling

Site-directed thiol cross-linking indicates that the first periplasmic loop (loop I/II) in the lactose permease of Escherichia coli is in close proximity to loops VII/VIII and XI/XII [Sun, J., and Kaback, H. R. (1997) Biochemistry 36, 11959-11965]. To determine whether thiol cross-linking reflects proximity as opposed to differences in the reactivity and/or dynamics of the Cys residues that undergo cross-linking, single-Cys mutants in loops I/II, VII/VIII, and XI/XII and double-Cys mutants in loop I/II and VII/VIII or XI/XII were purified and labeled with a sulfhydryl-specific nitroxide spin label. The labeled mutants were then analyzed by electron paramagnetic resonance (EPR) spectroscopy, and interspin distance was estimated from the extent of line shape broadening in the double-labeled proteins. Out of six paired double-Cys mutants that exhibit thiol cross-linking, five display significant spin-spin interaction. Furthermore, there is a qualitative correlation between distances estimated by site-directed cross-linking and EPR. Taken as a whole, the results are consistent with the conclusion that site-directed thiol cross-linking is primarily a reflection of proximity.     

Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum

The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca(2+) pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach. Leu(31) of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca(2+)-ATPase inhibition. We observed that L31A or L31C mutations of PLB prevented the inhibition of Ca(2+)-ATPase activity and disabled the cross-linking of N27C and N30C of PLB to Lys(328) and Cys(318) of SERCA2a. Although L31C-PLB failed to cross-link to any Cys or Lys residue of wild-type SERCA2a, L31C did cross-link with high efficiency to T317C of SERCA2a with use of the homobifunctional sulfhydryl cross-linking reagent, 1,6-bismaleimidohexane. This places Leu(31) of PLB within 10 angstroms of Thr(317) of SERCA2a in the M4 helix. Thus, contrary to previous suggestions, PLB with loss-of-function mutations at Leu(31) retains the ability to bind to SERCA2a, despite losing inhibitory activity. Cross-linking of L31C-PLB to T317C-SERCA2a occurred only in the absence of Ca(2+) and in the presence of nucleotide and was prevented by thapsigargin and by anti-PLB antibody, demonstrating for a fourth cross-linking pair that PLB interacts near M4 only when the Ca(2+) pump is in the Ca(2+)-free, nucleotide-bound E2 conformation, but not in the E2 state inhibited by thapsigargin. L31I-PLB retained full functional and cross-linking activity, suggesting that a bulky hydrophobic residue at position 31 of PLB is essential for productive interaction with SERCA2a. A model for the three-dimensional structure of the interaction site is proposed.     

Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation of the antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na⁺/H⁺ antiporter. However, because NhaA is active at physiological pH (pH 6.5-8.5), many questions related to the active state of NhaA have remained unanswered. Our Cys scanning of the highly conserved transmembrane VIII at physiological pH reveals that (1) the Cys replacement G230C significantly increases the apparent K_m of the antiporter to both Na⁺ (10-fold) and Li⁺ (6-fold). (2) Variants G223C and G230C cause a drastic alkaline shift of the pH profile of NhaA by 1 pH unit. (3) Residues Gly223-Ala226 line a periplasmic funnel at physiological pH as they do at pH 4. Both were modified by membrane-impermeant negatively charged 2-sulfonatoethyl methanethiosulfonate and positively charged 2-(trimethyl ammonium)-ethylmethanethiosulfonate sulfhydryl reagents that could reach Cys replacements from the periplasm via water-filled funnels only, whereas other Cys replacements on helix VIII were not accessible/reactive to the reagents. (4) Remarkably, the modification of variant V224C by 2-sulfonatoethyl methanethiosulfonate or 2-(trimethyl ammonium)-ethylmethanethiosulfonate totally inhibited antiporter activity, while N-ethyl maleimide modification had a very small effect on NhaA activity. Hence, the size—rather than the chemical modification or the charge—of the larger reagents interferes with the passage of ions through the periplasmic funnel. Taken together, our results at physiological pH reveal that amino acid residues in transmembrane VIII contribute to the cation passage of NhaA and its pH regulation.     

Cross-linking between helices within subunit a of Escherichia coli ATP synthase defines the transmembrane packing of a four-helix bundle

Subunit a of F(1)F(0) ATP synthase is required in the H(+) transport driven rotation of the c-ring of F(0), the rotation of which is coupled to ATP synthesis in F(1). The three-dimensional structure of subunit a is unknown. In this study, Cys substitutions were introduced into two different transmembrane helices (TMHs) of subunit a, and the proximity of the thiol side chains was tested via attempted oxidative cross-linking to form the disulfide bond. Pairs of Cys substitutions were made in TMHs 2/3, 2/4, 2/5, 3/4, 3/5, and 4/5. Cu(+2)-catalyzed oxidation led to cross-link formation between Cys pairs L120C(TMH2) and S144C(TMH3), L120C(TMH2) and G218C(TMH4), L120C(TMH2) and H245C(TMH5), L120C(TMH2) and I246C(TMH5), N148C(TMH3) and E219C(TMH4), N148C(TMH3) and H245C(TMH5), and G218C(TMH4) and I248C(TMH5). Iodine, but not Cu(+2), was found to catalyze cross-link formation between D119C(TMH2) and G218C(TMH4). The results suggest that TMHs 2, 3, 4, and 5 form a four-helix bundle with one set of key functional residues in TMH4 (Ser-206, Arg-210, and Asn-214) located at the periphery facing subunit c. Other key residues in TMHs 2, 4, and 5, which were concluded previously to compose a possible aqueous access pathway from the periplasm, were found to locate to the inside of the four-helix bundle.     

Increasing the refolding efficiency in vitro by site-directed mutagenesis of Cys383 in rat procarboxypeptidase B

This study examines a novel method to reduce the probability of disulfide mismatches during the refolding process by the replacement of cysteines within a protein. Specifically, Cys383 of recombinant rat procarboxypeptidase B was replaced by other amino acids to increase the refolding efficiency in vitro. Mutants C383G, C383A and C383S could refold successfully, but mutants C383R, C383E, C383L and C383Y failed to refold correctly. Compared with wild type, the refolding efficiencies of mutants C383G and C383A were enhanced. The Cys383 mutations changed some of the properties of rat carboxypeptidase B. Mutants C383G, C383A had higher k_cat/K_m values which indicated increased catalytic abilities. And both had higher thermal stability. pH had different effects on the activities and stabilities of the mutant and wild type proteins. The studies suggested that mutating Cys383 of rat procarboxypeptidase B could improve the renaturation process by increasing the refolding efficiency. This new method could be taken as a new attempt to improve the refolding efficiency of other recombinant proteins containing disulfide bonds that are expressed as inclusion bodies. While the results also claimed that the potential effects of the substituted amino acid on the protein itself should be seriously considered in addition to its ability to reduce the probability of disulfide mismatches.