Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.     

The orientation of transhydrogenase in the inner mitochondrial membrane of rat liver

The orientation of the transmembranous enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the nonspecific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Proteinase K treatment of mitoplasts converted the 110,000 transhydrogenase monomer into a single immunoreactive species having Mr 75,000. This proteolytic product is stable to further incubation with the protease. Treatment of submitochondrial particles with proteinase K resulted in the disappearance of the 110,000 monomer and the transient formation of an intermediate product with Mr 52,000. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. This model indicates that transhydrogenase (Mr 110,000) contains a core of proteolytically inaccessible proteins within the membrane (Mr 23,000) bounded by extramembranous domains on the matrix (Mr 52,000) and cytoplasmic (Mr 35,000) face of the inner mitochondrial membrane.     

The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini

The mitochondrial tricarboxylate carrier plays a fundamental role in the hepatic fatty acid synthesis. In this study, we investigated the transmembrane organization of this protein in the inner membrane of eel liver mitochondria using anti–N-terminal and anti–C-terminal antibodies. These antibodies recognized the N- and C-termini of the tricarboxylate carrier in intact mitoplasts, thus suggesting a cytosolic exposure of these regions in the membrane-bound protein. This structural arrangement of the tricarboxylate carrier was further confirmed by protease treatment of intact mitoplasts. Moreover, the oligomeric state of the native tricarboxylate carrier was investigated by blue native electrophoresis. A dimeric form of the carrier protein was found when eel liver mitochondria were solubilized with the mild detergent digitonin. These findings suggest an arrangement of the dimeric tricarboxylate carrier into an even number of membrane-spanning domains, with the N-terminal and C-terminal regions oriented toward the intermembrane space of fish mitochondria.     

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Intramitochondrial fatty acylation of a cytoplasmic imported protein in animal cells

Mitochondrial digitonin particles from mouse liver (and also from other tissues) incorporate [3H]myristic acid into a 52-kilodalton (kDa) protein in an energy-dependent manner. The 52-kDa N-myristylated protein is located inside the mitochondrial inner membrane since it is protected against proteolytic degradation in intact mitoplasts. Disruption of mitochondrial inner membrane by sonication results in severalfold higher labeling of the 52-kDa protein, further confirming that the enzyme system for protein fatty acylation as well as the 52-kDa target protein are compartmentalized inside the mitochondrial inner membrane matrix. The results of in vitro labeling of submitochondrial fractions suggest that both the 52-kDa target protein and the enzyme system for fatty acylation are in the matrix fraction, although the N-myristylated protein is found loosely associated with the inner membrane. Finally, immunoprecipitation of cytoplasmic free polysome translation products and in vitro transport of proteins into isolated mitochondria show that the 52-kDa protein is of cytoplasmic translation origin. These results demonstrate that the intramitochondrial N-myristylation of the 52-kDa protein is not translationally linked.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.     

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria.

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained. It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.     

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.     

Mammalian DNA polymerase beta: characterization of a 16-kDa transdomain fragment containing the nucleic acid-binding activities of the native enzyme.

The 39-kDa DNA polymerase beta (beta-Pol) molecule can be readily converted into two constituent domains by mild proteolysis; these domains are represented in an 8-kDa N-terminal fragment and a 31-kDa C-terminal fragment [Kumar et al. (1990a) J. Biol. Chem. 265, 2124-2131]. Intact beta-Pol is a sequence-nonspecific nucleic acid-interactive protein that binds both double-stranded (ds) and single-stranded (ss) polynucleotides. These two activities appear to be contributed by separate portions of the enzyme, since the 31-kDa domain binds ds DNA but not ss DNA, and conversely, the 8-kDa domain binds ss DNA but not ds DNA [Casas-Finet et al. (1991) J. Biol. Chem. 266, 19618-19625]. Truncation of the 31-kDa domain at the N-terminus with chymotrypsin, to produce a 27-kDa fragment (residues 140-334), eliminated all DNA-binding activity. This suggested that the ds DNA-binding capacity of the 31-kDa domain may be carried in the N-terminal segment of the 31-kDa domain. We used CNBr to prepare a 16-kDa fragment (residues 18-154) that spans the ss DNA-binding region of the 8-kDa domain along with the N-terminal portion of the 31-kDa domain. The purified 16-kDa fragment was found to have both ss and ds polynucleotide-binding capacity. Thermodynamic binding properties for these activities are similar to those of the intact enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).     

New outer membrane-associated protease of Escherichia coli K-12.

The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced. The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence. Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT. OmpP possessed the same enzymatic activity as OmpT. Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface. Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected. It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity. Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system.     

Studies on the structure of NADH: ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur protein component

Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of Mr 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490-496]. IP contains six subunits of Mr 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts.     

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained.It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.     

Lateral diffusion of redox components in the mitochondrial inner membrane is unaffected by inner membrane folding and matrix density

We report the first lateral diffusion measurements of redox components in normal-sized, matrix-containing, intact mitoplasts (inner membrane-matrix particles). The diffusion measurements were obtained by submicron beam fluorescence recovery after photobleaching measurements of individual, intact, rat liver mitoplasts bathed in different osmolarity media to control the matrix density and the extent of inner membrane folding. The data reveal that neither the extent of mitochondrial matrix density nor the complexity of the inner membrane folding have a significant effect on the mobility of inner membrane redox components. Diffusion coefficients for Complex I (NADH:ubiquinone oxidoreductase), Complex III (ubiquinol: cytochrome c oxidoreductase), Complex IV (cytochrome oxidase), ubiquinone, and phospholipid were found to be effectively invariant with the matrix density and/or membrane folding and essentially the same as values we reported previously for spherical, fused, ultralarge, matrix-free, inner membranes. Diffusion of proton-transporting Complex V (ATP synthase) appeared to be 2-3-fold slower at the greatest matrix density and degree of membrane folding. Consistent with a diffusion-coupled mechanism of electron transport, comparison of electron transport frequencies (productive collisions) with the theoretical, diffusion-controlled, collision frequencies (maximum collisions possible) revealed that there were consistently more calculated than productive collisions for all redox partners. Theoretical analyses of parameters for submicron fluorescence recovery after photobleaching measurements in intact mitoplasts support the finding of highly mobile redox components diffusing at the same rates as determined in conventional fluorescence recovery after photobleaching measurements in fused, ultralarge inner membranes. These findings support the Random Collision Model of Mitochondrial Electron Transport at the level of the intact mitoplast and suggest a similar conclusion for the intact mitochondrion.     

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol--cytochrome-c reductase complex.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.     

Biosynthesis of rat liver transhydrogenase in vivo and in vitro

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.     

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.     

Precise elimination of the N-terminal domain of histone H1.

The proteinase from mouse submaxillary gland was used to cleave total calf thymus histone H1 between residues 32 and 33. The C-terminal peptide, comprising residues 33 to the C-terminus, was purified and identified by amino acids analysis and Edman degradation. Spectroscopic characterization by n.m.r. for tertiary structure and by c.d. for secondary structure shows the globular domain of the parent histone H1 to be preserved intact in the peptide. It has therefore lost only the N-terminal domain and is a fragment of histone H1 comprising the globular plus C-terminal domains only. Precise elimination of only the N-terminal domain makes the fragment suitable for testing domain function in histone H1.     

Characterization of the heparin binding sites in human apolipoprotein E

Apolipoprotein (apo) E mediates lipoprotein remnant clearance via interaction with cell-surface heparan sulfate proteoglycans. Both the 22-kDa N-terminal domain and 10-kDa C-terminal domain of apoE contain a heparin binding site; the N-terminal site overlaps with the low density lipoprotein receptor binding region and the C-terminal site is undefined. To understand the molecular details of the apoE-heparin interaction, we defined the microenvironments of all 12 lysine residues in intact apoE3 and examined their relative contributions to heparin binding. Nuclear magnetic resonance measurements showed that, in apoE3-dimyristoyl phosphatidylcholine discs, Lys-143 and -146 in the N-terminal domain and Lys-233 in the C-terminal domain have unusually low pK(a) values, indicating high positive electrostatic potential around these residues. Binding experiments using heparin-Sepharose gel demonstrated that the lipid-free 10-kDa fragment interacted strongly with heparin and a point mutation K233Q largely abolished the binding, indicating that Lys-233 is involved in heparin binding and that an unusually basic lysine microenvironment is critical for the interaction with heparin. With lipidated apoE3, it is confirmed that the Lys-233 site is completely masked and the N-terminal site mediates heparin binding. In addition, mutations of the two heparin binding sites in intact apoE3 demonstrated the dominant role of the N-terminal site in the heparin binding of apoE even in the lipid-free state. These results suggest that apoE interacts predominately with cell-surface heparan sulfate proteoglycans through the N-terminal binding site. However, Lys-233 may be involved in the binding of apoE to certain cell-surface sites, such as the protein core of biglycan.