Unique features of the pre-T-cell receptor alpha-chain: not just a surrogate

The pre-T-cell receptor (pre-TCR) has a crucial role in the normal development of alphabeta T cells. Different views have emerged concerning the structure and function of the pre-TCR. This molecular complex can be viewed as a variant of the alphabeta-TCR in which the pre-TCR alpha-chain that is covalently associated with the TCR beta-chain is a 'surrogate' TCR alpha-chain. Alternatively, the unique structure of the pre-TCR might be associated with a unique function, owing to evolutionary selection of a pre-TCR alpha-chain that has different capabilities from the TCR alpha-chain. As described here, I consider that experimental evidence favours the latter view.     

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.     

The alpha-chain-termination mutants and their relation to the alpha-thalassaemias.

The structure, synthesis, genetic transmission, clinical associations and distribution of the elongated alpha-chain haemoglobin variants has been described. The data indicate that the most likely molecular basis for these common abnormal haemoglobins is a single base substitution in the alpha-chain termination codon. Because these variants are produced inefficiently they give rise to the clinical picture of alpha-thalassaemia. When these findings are taken together with recent work regarding the molecular basis for other forms of alpha-thalassaemia it is possible to build up a fairly complete picture of the molecular pathology of the alpha-thalassaemias.     

LCAT activation properties of apo A-I CNBr fragments and conversion of discoidal complexes into spherical particles.

We studied the substrate properties of the phospholipid-cholesterol-apolipoprotein complexes generated with apo A-I, apo A-I-CNBr fragments, apo A-II and apo A-IV for cholesterol esterification by the enzyme lecithin-cholesterol acyltransferase (LCAT). The kinetic parameters determined with the different complexes as substrates, showed that the complexes containing apo A-I and apo A-IV were about 40-times more efficient than those generated with the apo A-I fragments. In this system, the substrates containing apo A-II had the lowest efficiency. In spite of the differences in the kinetic parameters observed with the various apolipoprotein-lipid complexes, the cholesterol inserted in the complexes was esterified for more than 90% after 24 h in all systems studied. Based upon the results of the kinetic experiments, we followed the transformation of the discoidal complexes into spherical particles, due to the formation of a cholesteryl esters core, in the presence of low-density lipoproteins as an external source of cholesterol. We observed the formation of spherical particles by electron microscopy, after incubation of the discoidal complexes with LCAT for 24 h. The average percentage of cholesteryl esters in the converted particles was around 60% of the total cholesterol, varying between 40% for the apo A-I-CNBr-1-DPPC-cholesterol complex and up to 86% for the apo A-I-DPPC-cholesterol complex. The secondary structure of protein in the complexes was not significantly modified. However, the phospholipid phase transition disappeared, together with the parallel orientation of the phospholipid acyl chains with the helical segments of the apolipoproteins, as the phospholipids are organized in a monolayer at the surface of the spheres.     

Structure of the apo form of Bacillus stearothermophilus phosphofructokinase

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 ? resolution (Protein Data Bank entry 3U39 ). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the alpha-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet alpha-chain and that this approach should be similarly effective in HbA.     

Localization of the alpha-chain cross-link acceptor sites of human fibrin

The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain.     

Structural and functional role of nickel ions in urease by molecular dynamics simulation

Nickel ions play several roles in the biological processes of microorganisms and plants. Urease has a nickel-containing active site and catalyzes the hydrolysis of urea to yield ammonia and carbamate. In the present study, the role of nickel ions is examined using molecular dynamics simulations of the holo and apo forms. Nonbonded models used for the nickel ions provide good reproduction of the active-site structure as indicated in the crystallized structure. The results confirm that urease has a rigid active site in either its holo or its apo form. A new conformation of the flap is observed in apo urease. The connection between the metal center and His^α323 is proposed to be responsible for maintaining the flap conformation. The binding free energy of acetohydroxamic acid and urease is estimated using the molecular mechanics–generalized Born/surface area method. The binding free energy is primarily driven by electrostatic interactions in the presence of nickel ions. Normal mode analysis is employed to characterize the movements of the flap in apo urease.     

Apo alpha-lactalbumin and lysozyme are colocalized in their subsequently formed spherical supramolecular assembly

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.     

Modeling of the metallo-beta-lactamase from B. fragilis: structural and dynamic effects of inhibitor binding

The structure and dynamics of an inhibitor-bound complex of the metallo-beta-lactamase from Bacteroides fragilis are studied by using molecular dynamics. A search of the conformational space was performed to obtain three distinct models of the complex, which were then subjected to solvated molecular dynamics. A solvated molecular dynamics study of the apo protein was performed to serve as a baseline for comparison with the bound simulations. We find loop conformation changes due to binding as well as a decrease in flexibility of the protein as a whole and especially in the major loop of the beta-lactamase. We report the structural and dynamical features of the inhibitor-bound and apo models, as well as experimentally measurable quantities, which should be capable of distinguishing the two binding modes we have determined.     

Permissible discontinuity region of the alpha-chain of hemoglobin: noncovalent interaction of heme and the complementary fragments alpha 1-30 and alpha 31-141

Generation of a fragment-complementing system of the alpha-chain on limited proteolysis with Staphylococcus aureus V8 protease has been investigated. Digestion of the alpha-chain (0.4 mM) of hemoglobin with V8 protease in phosphate buffer at pH 6.0 and 37 degrees C is limited to the peptide bonds of Glu-23, Glu-27, Glu-30, and Asp-47. Gel filtration of a V8 protease digest of the alpha-chain on a Sephadex G-50 column did not release any heme to the low molecular weight region, though some peptides were released from the protein. The filtration studies revealed the presence of two heme-containing components in the digest, the major one eluting at the alpha-chain position and the minor one eluting slightly ahead of the alpha-chain position. Reversed-phase high-performance liquid chromatography and amino-terminal sequence analysis demonstrated that the component eluting at the alpha-chain position contains species generated by the noncovalent interactions of heme and the complementary fragments alpha 1-30 and alpha 31-141. In dilute solutions (0.04 mM) the V8 protease digestion occurred exclusively on the carboxyl side of Glu-30(alpha). This high selectivity was also observed at pH 4.0 and pH 7.8. The visible spectra and the ultraviolet circular dichroic spectra of the digest reflect the native-like structure of the noncovalent fragment system. The dissociation constant of alpha 1-30 appears to be in the range of 10(-8) M. In tetrameric hemoglobin A the peptide bond of Glu-30-Arg-31 of the alpha-chain is not accessible to V8 protease digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase

Structures have been determined of Bacillus stearothermophilus "apo" and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. The "apo" lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"-enzyme. Further refinement proceeded with the isomorphous holo-enzyme from Bacillus stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--one at the substrate binding site and one close to the molecular P-axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo" model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with nicotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose 1,6-bisphosphate were also analyzed.     

Antibodies to synthetic peptides as probes of acetylcholine receptor structure

Peptides containing the C-terminus of the alpha-chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide-specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alpha-chain. Trypsinization experiments and the use of the unique C-terminal hexapeptide of the alpha-chain demonstrated that the proposed C-terminus does exist on the mature alpha-chain, and that post-translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha-chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa). Cleavage of the alpha-chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9-10 kDa). No signs of experimental myasthenia gravis were observed in rabbits immunised with C-terminal peptide coupled to carrier protein.     

Isolation and characterization of the two major apoproteins in human lipoprotein [a]

Human Lp[a] was isolated in preparative amounts from two donors; the native lipoprotein and its constituent apoproteins, apo[a] and apoB, were characterized extensively. Based on differences in apparent molecular weight, four different isoforms of apo[a], a1-a4, were observed between the two donors. The number and relative distribution of these isoforms varied between donors but were constant for each donor. Each apo[a] isoform was shown to be derived from a discrete apo[a]-B100 disulfide-linked complex present before reduction. Complete delipidation of Lp[a] was followed by solubilization, reduction, and carboxamidomethylation of the constituent apoproteins. These apoproteins were then separated by immunoaffinity chromatography using anti-apo[a]- or anti-apoB-Sepharose; their purity and structural integrity were demonstrated by Western blot analysis. ApoB isolated by this procedure was essentially identical to apoB from autologous LDL with respect to molecular weight, secondary structure, amino acid composition, and sialic acid content. However, apo[a] differed from apoB in that it exhibited: a much less alpha-helical, less beta, but much more disordered structure; a lower proportion of aspartate, isoleucine, leucine, phenylalanine, and lysine, but a higher proportion of proline, glycine, and threonine; and a much higher content of sialic acid. These results indicate that apo[a] is not a superglycosylated form of apoB but is distinctly different in its composition and structure.     

The spectrin super-family

The review is focused on recent data on the primary sequences of erythroid and non-erythroid spectrins. As in other fields, the techniques of molecular genetics have allowed great advances in our knowledge of the structure and the genetic story of these molecules. Comparison of alpha-chains sequences of the non-erythroid (fodrin) and erythroid spectrin demonstrated that the fodrin alpha-genes are strictly conserved across species, while the mammalian spectrin genes have diverged rapidly. Spectrin and fodrin alpha-chains are largely composed of homologous 106-amino-acid repeat units. Spectrin alpha-chain is lacking a 37 amino-acid sequence which bears the calmodulin-binding site of the fodrin alpha-chain. The highest degree of homology between the spectrin alpha-chain and the fodrin alpha-chain lies in a central atypical segment unrelated to the canonical repeat sequence. This region is closely related to the N-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. Like the spectrin alpha-chain, the major central part of the spectrin beta-chain is made up of repeat units of 106 amino-acids. The N-terminal domain of the beta-chain, and especially the actin binding site, is the region of greatest homology among members of the spectrin super-family, including Drosophila spectrin beta-chain, dystrophin and alpha-actinin. The C-terminal extremity of the erythroid beta-chain is also of great interest, since tissue-specific differential processing of 3'beta-spectrin gene pre-mRNA generates a beta spectrin-isoform with a unique C-terminus in human skeletal muscle.     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

The molecular organization of the protein HC-IgA complex (HC-IgA)

Complexes of protein HC and monoclonal IgA1 or IgA2 or polyclonal IgA were isolated from human blood plasma. Dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that all complexes contain three types of chains: two light immunoglobulin chains, one regular IgA alpha-chain, and one chain with Mr = 90,000 carrying both alpha-chain and protein HC epitopes. The complexes were split into Fab alpha and Fc alpha fragments by bacterial IgA proteases. The protein HC epitopes were linked to the Fc fragments. Complexes of protein HC and an alpha-chain devoid of the variable region and the first heavy chain constant domain could also be demonstrated to be present in the blood plasma of a patient with alpha-heavy chain disease. Pepsin digestion of HC-IgA released a fragment containing all the protein HC epitopes and the C-terminal nonapeptide of the IgA alpha-chain. The light immunoglobulin chains, the regular alpha-chain, and the 90,000-Da chain from monoclonal HC-IgA1 were isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis and by repeated gel filtration in dodecyl sulfate-containing buffer. The N-terminal amino acid sequence of the alpha-chain was identical with that of a regular human heavy immunoglobulin chain of subgroup III. Subtractive degradations of the 90,000-Da chain displayed 2 amino acid residues in each position in a pattern suggesting simultaneous degradations of a chain identical with the regular alpha-chain of HC-IgA and of uncomplexed, low molecular weight, protein HC. All the results are compatible with a model for HC-IgA in which a single low molecular weight protein HC polypeptide chain is covalently linked, side by side, to the C-terminal nonapeptide of one of the two alpha-chains of a regular monomeric IgA unit.     

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.     

Computational and experimental approaches for assessing the interactions between the model calycin beta-lactoglobulin and two antibacterial fluoroquinolones

Norfloxacin and levofloxacin, two fluoroquinolones of different bulk, rigidity and hydrophobicity taken as model ligands, were docked to one apo and two holo crystallographic structures of bovine beta-lactoglobulin (BLG) using different computational approaches. BLG is a member of the lipocalin superfamily. Lipocalins show a typical b-barrel structure encompassing an internal cavity where small hydrophobic molecules are usually bound. Our studies allowed the identification of two putative binding sites in addition to the calyx. The rigid docking approximation resulted in strong repulsive forces when the ligands were docked into the calyx of the apo form. On the contrary, hindrance was not experienced in flexible docking protocols whether on the apo or on the holo BLG forms, due to allowance for side chain rearrangement. K(i) between 10(-7) and 10(-6) M were estimated for norfloxacin at pH 7.4, smaller than 10(-5) M for levofloxacin. Spectroscopic and electrophoretic techniques experimentally validated the occurrence of an interaction between norfloxacin and BLG. Changes in chemical shift and dynamic parameters were observed between the (19)F NMR spectra of the complex and of the ligand. A K(i) (ca 10(-7) M) comparable with the docking results was estimated through a NMR relaxation titration. Stabilization against unfolding was demonstrated by denaturant gradient gel electrophoresis on the complex versus apo BLG. NMR experimental evidence points to a very loose interaction for ofloxacin, the racemic mixture containing levofloxacin. Furthermore, we were able to calculate in silico K(i)'s comparable to the published experimental values for the complexes of palmitic and retinoic acid with BLG.