Structural, bioinformatic, and in vivo analyses of two Treponema pallidum lipoproteins reveal a unique TRAP transporter

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of "tetratricopeptide repeat" (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).     

Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.     

Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques,site directed mutagenesis,circular dichroism and ligand binding assays

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.     

Interactions among the A and T units of an ECF-type biotin transporter analyzed by site-specific crosslinking

Energy-coupling factor (ECF) transporters are a huge group of micronutrient importers in prokaryotes. They are composed of a substrate-specific transmembrane protein (S component) and a module consisting of a moderately conserved transmembrane protein (T component) and two ABC ATPase domains (A components). Modules of A and T units may be dedicated to a specific S component or shared by many different S units in an organism. The mode of subunit interactions in ECF transporters is largely unknown. BioMNY, the focus of the present study, is a biotin transporter with a dedicated AT module. It consists of the S unit BioY, the A unit BioM and the T unit BioN. Like all T units, BioN contains two three-amino-acid signatures with a central Arg residue in a cytoplasmic helical region. Our previous work had demonstrated a central role of the two motifs in T units for stability and function of BioMNY and other ECF transporters. Here we show by site-specific crosslinking of pairs of mono-cysteine variants that the Ala-Arg-Ser and Ala-Arg-Gly signatures in BioN are coupling sites to the BioM ATPases. Analysis of 64 BioN-BioM pairs uncovered interactions of both signatures predominantly with a segment of ~13 amino acid residues C-terminal of the Q loop of BioM. Our results further demonstrate that portions of all BioN variants with single Cys residues in the two signatures are crosslinked to homodimers. This finding may point to a dimeric architecture of the T unit in BioMNY complexes.     

Extramembrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport

We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. (2002) Nature 419, 587-593). The extramembrane headpiece of the AcrB trimer contains a central pore composed of three alpha-helices. Each pore helix belongs to a different monomer. In this study, we constructed cysteine-scanning mutants as to the residues comprising the pore helix. Of the 21 mutants (D99C to P119C), 5 (D101C, V105C, N109C, Q112C, and P116C) showed significantly reduced drug resistance and drug-exporting activity. These residues are localized on one side of the pore helix, i.e. on the wall of the pore. These observations strongly indicate the important role of this pore in the drug transport process. A N-ethylmaleimide binding experiment revealed that the pore is in the closed state, and the thickness of the permeability barrier in the middle of the pore corresponds to 2.5 alpha-helical turns. Two mutants (V105C and Q112C), which showed the greatest loss of activity of all of the pore mutants, were detected in the form of disulfide cross-linking dimers under normal conditions, suggesting that a conformational change of the pore is indispensable during the transport process.     

Trivalent recognition unit of innate immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like domain

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9A resolution. Although the FD1 monomer shares a common fold with the fibrinogen gamma fragment and tachylectin-5A, the Asp-282-Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-d-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282-Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self- and non-self discrimination mechanism by ficolins.     

Structural and biochemical basis for polyamine binding to the Tp0655 lipoprotein of Treponema pallidum: putative role for Tp0655 (TpPotD) as a polyamine receptor

Tp0655 of Treponema pallidum, the causative agent of syphilis, is predicted to be a 40 kDa membrane lipoprotein. Previous sequence analysis of Tp0655 noted its homology to polyamine-binding proteins of the bacterial PotD family, which serve as periplasmic ligand-binding proteins of ATP-binding-cassette (ABC) transport systems. Here, the 1.8 A crystal structure of Tp0655 demonstrated structural homology to Escherichia coli PotD and PotF. The latter two proteins preferentially bind spermidine and putrescine, respectively. All of these proteins contain two domains that sandwich the ligand between them. The ligand-binding site of Tp0655 can be occupied by 2-(N-morpholino)ethanesulfanoic acid, a component of the crystallization medium. To discern the polyamine binding preferences of Tp0655, the protein was subjected to isothermal titration calorimetric experiments. The titrations established that Tp0655 binds polyamines avidly, with a marked preference for putrescine (Kd=10 nM) over spermidine (Kd=430 nM), but the related compounds cadaverine and spermine did not bind. Structural comparisons and structure-based sequence analyses provide insights into how polyamine-binding proteins recognize their ligands. In particular, these comparisons allow the derivation of rules that may be used to predict the function of other members of the PotD family. The sequential, structural, and functional homology of Tp0655 to PotD and PotF prompt the conclusion that the former likely is the polyamine-binding component of an ABC-type polyamine transport system in T. pallidum. We thus rename Tp0655 as TpPotD. The ramifications of TpPotD as a polyamine-binding protein to the parasitic strategy of T. pallidum are discussed.     

Structural characterization of a mannose-binding protein-trimannoside complex using residual dipolar couplings

The ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) have been investigated using residual dipolar couplings (RDCs) that are easily measured from NMR spectra of the ligand and isotopically labeled protein. Using a limited set of 1H-15N backbone amide NMR assignments for MBP and orientational information derived from the RDC measurements in aligned media, an order tensor for MBP has been determined that is consistent with symmetry-based predictions of an axially symmetric system. 13C-1H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (trimannoside) have been determined at natural abundance and used as orientational constraints. The bound ligand geometry and orientational constraints allowed docking of the trimannoside ligand in the binding site of MBP to produce a structural model for MBP-oligosaccharide interactions.     

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.     

Transport of nickel and cobalt ions into bacterial cells by S components of ECF transporters

Energy-coupling factor (ECF) transporters form a distinct group of ABC-type micronutrient importers in prokaryotes that do not contain extracytoplasmic, soluble substrate-binding proteins. Instead, they consist of a transmembrane substrate-specific S component that interacts with a module composed of a moderately conserved transmembrane (T) component and ABC ATPases. The majority of S components is considered to act as high-affinity binding proteins that strictly depend on their cognate T and ATPase units for transport activity. For a fraction of biotin-specific S units, however, transport activity was demonstrated in their solitary state. Here, we compared the activities of nickel- and cobalt-specific ECF transporters in the presence and absence of their T and ATPase units. Accumulation assays with radioactive metal ions showed that the truncated transporters led to approx. 25 % of cell-bound radioactivity compared to the holotransporters. Activity of urease, an intracellular nickel-dependent enzyme, was used as a reporter and clearly indicated that the cell-bound radioactivity correlates with the cytoplasmic metal concentration. The results demonstrate that S units of metal transporters not only bind their substrates on the cell surface but mediate transport across the membrane, a finding of general importance on the way to understand the mechanism of ECF transporters.     

Structural rearrangements at the translocation pore of the human glutamate transporter, EAAT1

Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-beta-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport.     

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an open-unliganded to closed-liganded state during the transport cycle.     

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.     

Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization

The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Histidine-regulated activity of M-ficolin

Human M-ficolin is a pathogen-associated molecular recognition molecule in the innate immune system, and it binds to some sugars, such as GlcNAc (N-acetylglucosamine), on pathogen surfaces. From previous structural and functional studies of the FD1 (M-ficolin fibrinogen-like domain), we proposed that the ligand-binding region of FD1 exists in a conformational equilibrium between active and non-active states depending on three groups with a pK(a) of 6.2, which are probably histidine residues, and suggested that the 2-state conformational equilibrium as well as the trimer formation contributes to the discrimination mechanism between self and non-self of FD1 [Tanio, M., Kondo, S., Sugio, S. and Kohno, T. (2007) J. Biol. Chem. 282, 3889-3895]. To investigate the origins of the pH dependency, mutational analyses were performed on FD1 expressed by Brevibacillus choshinensis. The GlcNAc binding study of a series of single histidine mutants of FD1 demonstrated that His(251), His(284) and His(297) are required for the activity, and thus we concluded that the three histidines are the origins of the pH dependency of FD1. Monomeric mutants of FD1 show weaker affinity for the ligand than the trimeric wild-type, indicating that trimer formation confers high avidity for the ligand. In addition, analyses of the GlcNAc association and dissociation of FD1 provided evidence that FD1 always exchanges between the active and non-active states with the pH-dependent populations in solution. The biological roles of the histidine-regulated conformational equilibrium of M-ficolin are discussed in terms of the self and non-self discrimination mechanism.     

Glycosylation of the OCTN2 carnitine transporter: Study of natural mutations identified in patients with primary carnitine deficiency

Primary carnitine deficiency is caused by impaired activity of the Na⁺-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.     

Charge stabilization in reaction center protein investigated by optical heterodyne detected transient grating spectroscopy

Photosynthetic reaction center (RC) pigment protein complex converts the free energy of light into chemical potential of charge pairs with extremely high efficiency. A transient phase in the absorption spectrum in the sub-millisecond time scale is expected to be especially important to examine the conformational gating model of the Q (A) (-) Q(B) to Q(A)Q (B) (-) (here Q(A )and Q(B) are the primary and secondary quinone type electron acceptors, respectively) electron transport. Essential kinetic components at few tens of microseconds scale and at around 200 mus have been suggested. We investigated the conformation change of RCs using heterodyne detection of the laser-induced transient grating method. An about 25 mus dynamics was observed, which coincides with the one described by the conformational gating model and possibly related to the nonadiabatic intrinsic Q (A) (-) Q(B) to Q(A)Q (B) (-) electron transport. The relative intensity of this component decreased with increasing quinone concentration indicating an initial (P(+)Q (A) (-) )(1) or a relaxed (P(+)Q (A) (-) )(2 )conformational substate. We did not find the decay component at few hundreds of microseconds time scale indicating that there is no large displacement in the RC structure if Q(B) is present. The diffusion coefficient of the RC/LDAO detergent micelles calculated from the kinetic component was D = 3.8 x 10(-11 )m(2)/s that agrees fairly well with the number estimated from the Einstein-Stokes relationship, and relates to a hydrodynamic diameter of 11.4 nm of the RC in LDAO micellar solution.     

Oligomeric structure of the carnitine transporter CaiT from Escherichia coli

The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.     

Identification of Porphyromonas gingivalis components that mediate its interactions with fibronectin.

Porphyromonas (Bacteroides) gingivalis W12 binds and degrades human plasma fibronectin. In the presence of the protease inhibitor N-alpha-p-tosyl-L-lysyl chloromethyl ketone, P. gingivalis cells accumulated substantial amounts of 125I-fibronectin as a function of incubation time. Fibronectin binding was specific, reversible, and saturable. The Kd for the reaction was estimated to be on the order of 100 nM, and there was an average of 3.5 x 10(3) fibronectin binding sites per cell. Unlabeled fibronectin inhibited the binding of 125I-fibronectin to bacteria; however, fibrinogen was an even more efficient inhibitor of 125I-fibronectin binding. Unrelated proteins were without effect on fibronectin binding. A fibronectin-binding component (Mr, 150,000) was identified in sodium dodecyl sulfate-solubilized P. gingivalis. Fibronectin was degraded into discrete peptides by P. gingivalis W12. The degradation of fibronectin was inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone. Two P. gingivalis components (Mrs, 120,000 and 150,000) degraded fibronectin in substrate-containing gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a previous study (M. S. Lantz, R. D. Allen, T. A. Vail, L. M. Switalski, and M. Hook, J. Bacteriol. 173:495-504, 1991), we found that the same strain of P. gingivalis bound and subsequently degraded human fibrinogen via apparently distinct cell surface components of molecular sizes similar to those of components now implicated in the binding and degradation of fibronectin. These results raise the possibility that the two ligands are recognized and modified by the same components on P. gingivalis W12. In support of this hypothesis, unlabeled fibrinogen effectively inhibited the binding of 125I-fibronectin to bacteria and blocked 125I-fibronectin binding to a P. gingivalis ligand-binding component (Mr, 150,000 immobilized on a nitrocellulose membrane.