Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity.

Platelet glycoprotein (GP) Ibalpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of approximately 300 residues of GPIbalpha mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbalpha, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibalpha to these ligands.

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs.

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.

Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin.

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.

Interaction of von Willebrand factor domain A1 with platelet glycoprotein Ibalpha-(1-289). Slow intrinsic binding kinetics mediate rapid platelet adhesion.

We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibalpha. Recombinant VWF A1 domain (residues Glu(497)-Pro(705) of VWF) bound stoichiometrically to a GPIbalpha-calmodulin fusion protein (residues His(1)-Val(289) of GPIbalpha; GPIbalpha-CaM) immobilized on W-7-agarose with a K(d) of 3.3 microM. The variant VWF A1(R545A) bound to GPIbalpha-CaM 20-fold more tightly, mainly because the association rate constant k(on) increased from 1,100 to 8,800 M(-1) s(-1). The GPIbalpha mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIbalpha(G233V)-CaM and GPIbalpha(M239V)-CaM with a K(d) of 1.0 and 0.63 microM, respectively. The increased affinity of VWF A1 for GPIbalpha(M239V)-CaM was explained by an increase in k(on) to 4,500 M(-1) s(-1). GPIbalpha-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu(480)/Val(481)-Gly(718)). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIbalpha-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIbalpha-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIbalpha, and the increased binding affinity caused by mutations in VWF or GPIbalpha, are reproduced by isolated structural domains. The substantial increase in k(on) caused by mutations in either A1 or GPIbalpha suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k(on) and k(off) provide important new constraints on models for rapid platelet tethering at high wall shear rates.

Threonine-145/methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbalpha express HPA-2 epitopes and show equal binding of von Willebrand factor.

Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIbalpha is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIbalpha were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIbalpha in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIbalpha in vWF binding and indicate that recombinant GPIbalpha may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)

The domain structure of human receptor-associated protein. Protease sensitivity and guanidine HCl denaturation.

The 39-kDa receptor-associated protein (RAP), a specialized chaperone for endocytic receptors of the low density lipoprotein receptor gene family, is a triplicate repeat sequence (residues 1-100, 101-200, and 201-323, respectively), with the three repeats having different functional roles. The goal of the present study was to use a combination of protease sensitivity and guanidine denaturation analyses to investigate whether human RAP correspondingly contained multiple structural domains. Protease sensitivity analysis using six proteolytic enzymes of varying specificity showed that RAP has two protease-resistant regions contained within repeat 1 (residues 15-94) and repeat 3 (residues 223-323). Guanidine denaturation analysis showed that RAP has two phases in its denaturation, an early denaturation transition at 0.6 M guanidine HCl, and a broad second transition between 1.0 and 3.0 M guanidine HCl. Analysis of the denaturation of the individual repeats showed that, despite the similarity in sequence and protease sensitivity between repeats 1 and 3, repeat 1 was a stable structure, with a sharp transition midpoint at 2.4 M guanidine HCl, while repeat 3 was relatively unstable, with a transition midpoint at 0.6 M guanidine HCl. Repeat 2 had a denaturation profile almost identical to that of repeat 3. Denaturation analysis of the contiguous repeats 1 and 2 (residues 1-210) indicated that repeats 1 and 2 probably interact to form one structural domain represented by the broad transition, while repeat 3 constitutes a separate domain represented by the early transition. A two-domain model of RAP three-dimensional structure is proposed that integrates both structural and functional information, in which a helical segment from repeat 2 interacts with the known three-helix bundle of repeat 1 to form a four-helix bundle structural domain, while repeat 3 forms the other structural domain.

Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1.

The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.

Differential functions of triplicated repeats suggest two independent roles for the receptor-associated protein as a molecular chaperone.

The 39-kDa receptor-associated protein (RAP) is a molecular chaperone for the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that binds multiple ligands. The primary function of RAP has been defined as promotion of the correct folding of LRP, and prevention of premature interaction of ligands with LRP within the early secretory pathway. Previous examination of the RAP sequence revealed an internal triplication. However, the functional implication of the triplicated repeats was unknown. In the current study using various RAP and LRP domain constructs, we found that the carboxyl-terminal repeat of RAP possesses high affinities to each of the three ligand-binding domains on LRP, whereas the amino-terminal and central repeats of RAP exhibit only low affinity to the second and the fourth ligand-binding domains of LRP, respectively. Using truncated soluble minireceptors of LRP, we identified five independent RAP-binding sites, two on each of the second and fourth, and one on the third ligand-binding domain of LRP. By coexpressing soluble LRP minireceptors and RAP repeat constructs, we found that only the carboxyl-terminal repeat of RAP was able to promote the folding and subsequent secretion of the soluble LRP minireceptors. However, when the ability of each RAP repeat to inhibit ligand interactions with LRP was examined, differential effects were observed for individual LRP ligands. Most striking, both the amino-terminal and central repeats, but not the carboxyl-terminal repeat, of RAP inhibited the interaction of alpha2-macroglobulin with LRP. These differential functions of the RAP repeats suggest that the roles of RAP in the folding of LRP and in the prevention of premature interaction of ligand with the receptor are independent.

The 2.6 A structure of antithrombin indicates a conformational change at the heparin binding site.

The crystal structure of a dimeric form of intact antithrombin has been solved to 2.6 A, representing the highest-resolution structure of an active, inhibitory serpin to date. The crystals were grown under microgravity conditions on Space Shuttle mission STS-67. The overall confidence in the structure, determined earlier from lower resolution data, is increased and new insights into the structure-function relationship are gained. Clear and continuous electron density is present for the reactive centre loop region P12 to P14 inserting into the top of the A-beta-sheet. Areas of the extended amino terminus, unique to antithrombin and important in the binding of the glycosaminoglycan heparin, can now be traced further than in the earlier structures. As in the earlier studies, the crystals contain one active and one latent molecule per asymmetric unit. Better definition of the electron density surrounding the D-helix and of the residues implicated in the binding of the heparin pentasaccharide (Arg47, Lys114, Lys125, Arg129) provides an insight into the change of affinity of binding that accompanies the change in conformation. In particular, the observed hydrogen bonding of these residues to the body of the molecule in the latent form explains the mechanism for the release of newly formed antithrombin-protease complexes into the circulation for catabolic removal.

Inhibitory mechanism of serpins. Mobility of the C-terminal region of the reactive-site loop.

The reactive-site loops of serpins are characterized by a defined mobility where the loop adopts a new secondary structure as an essential part of the inhibitory process. While the importance of mobility in the N-terminal region of the reactive-site loop has been well studied, the role of mobility in the C-terminal portion has not been investigated. The requirements for mobility of the C-terminal portion of the reactive-site loop of alpha1-antitrypsin were investigated by creating a disulfide bridge between the P'3 residue and residue 283 near the top of strand 2C; this disulfide would restrict the mobility of the C-terminal portion of the reactive-site loop by locking together strands 1 and 2 of the C beta-sheet. The engineered disulfide bond had no effect on the inhibitory activity of alpha1-antitrypsin, indicating that there is no requirement for mobility in this region of the molecule. Moreover, these results, coupled with those from molecular modeling, indicate that insertion into the A beta-sheet of the intact reactive-loop beyond P12 is not rate-limiting for the formation of the stable complex. The engineered disulfide bond should also prove useful in the creation of more stable serpin variants; for example, such a bond in plasminogen activator inhibitor-1 would prevent it from becoming latent by locking strand 1C onto the C beta-sheet.

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Improved diffraction of antithrombin crystals grown in microgravity.

Crystals of antithrombin were grown both on earth and in microgravity aboard US Space Shuttle Flight STS-67. The quality of crystals grown in both environments was highly variable and many could not be indexed. The microgravity crystals, however, generally diffracted better, as demonstrated by a novel procedure that estimates the resolution of the Bragg scatter from single diffraction images, without requiring knowledge of the cell dimensions of the crystal. Whereas the best earth-grown crystals never diffracted beyond 3 angstroms resolution, the best microgravity crystal diffracted to 2.6 angstroms. The improvement, demonstrated here by a comparison of 23 microgravity and 12 earth-grown crystals, is attributed to better ordered crystal growth in microgravity, although other factors may have contributed also.

Probing serpin reactive-loop conformations by proteolytic cleavage.

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the relationship of their reactive-loop conformations to those of circulating forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and anti-thrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop peptides. Proteolysis at the P10-P9, P8-P7 and P7-P6 of anti-trypsin was distorted by binary complex formation. The P1'-P2' bond in anti-thrombin was more accessible to proteolysis after binary complex formation, whereas cleavage at the P4-P3 bond was variably altered by synthetic peptide insertion. The proteolytic accessibility of the reactive-site P1-P1' bond of anti-trypsin and anti-thrombin binary complexes was identical with that of the native form and no cleavage was observed in the hinge region (P15-P10) of either protein, whether native or as binary complexes. these results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in the circulation, with the hinge region being partly incorporated into the A beta-pleated sheet. This loop can be displaced by peptides and induced to adopt a new conformation similar to the three-turn helix of ovalbumin. Both the native and binary complexed forms of anti-thrombin showed a greatly increased proteolytic sensitivity in the presence of heparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates the approximation of enzyme and inhibitor.

Preparation and characterization of latent alpha 1-antitrypsin.

Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from < 1% to approximately 35% by refolding from 6 M guanidinium chloride.

Thromboembolic disease due to thermolabile conformational changes of antithrombin Rouen-VI (187 Asn-->Asp)

A new variant of antithrombin (Rouen-VI, 187 Asn-->Asp) with increased heparin affinity was shown to have normal inhibitory activity which decreased slowly at 4 degrees C and rapidly at 41 degrees C. On electrophoresis the freshly isolated variant had an anodal shift relative to native antithrombin due to the mutation. A further anodal transition occurred after either prolonged storage at 4 degrees C or incubation at 41 degrees C due to the formation of a new inactive uncleaved component with properties characteristic of L-form (latent) antithrombin. At the same time, polymerization also occurred with a predominance of di-, tri-, and tetra-mers. These findings fit with the observed mutation of the conserved asparagine (187) in the F-helix destabilizing the underlying A-sheet of the molecule. Evidence of A-sheet perturbation is provided by the increased rate of peptide insertion into the A-sheet and by the decreased vulnerability of the reactive loop to proteolysis. The spontaneous formation of both L-antithrombin and polymers is consistent with our crystal structure of intact antithrombin where L-form and active antithrombin are linked together as dimers. The nature of this linkage favors a mechanism of polymerization whereby the opening of the A-sheet, to give incorporation of the reactive center loop, is accompanied by the bonding of the loop of one molecule to the C-sheet of the next. The accelerated lability of antithrombin Rouen-VI at 41 versus 37 degrees C provides an explanation for the clinical observation that episodes of thrombosis were preceded by unrelated pyrexias.

Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms.

Human apolipoprotein (apo) E4 (arginine at residue 112) preferentially associates with very low density lipoproteins (VLDL), and apoE3 (cysteine at 112) associates with high density lipoproteins. It has been postulated that the amino-terminal domain, which contains residue 112, influences the lipoprotein preference by interacting with the carboxyl-terminal domain, which contains the lipid-binding region. To delineate the region in the carboxyl-terminal domain mediating lipoprotein binding and involved in isoform preference, we produced truncated apoE3 and apoE4 variants (terminating at residues 251, 260, 266, or 272) in Escherichia coli and assessed them for lipoprotein association. This analysis suggested that residues 260-272 contain important determinants for complete lipoprotein association and isoform preferences. To determine whether positive charge at residue 112 was an absolute requirement for the apoE4 VLDL preference, we compared the distributions of rabbit apoE (equivalent to apoE3, with cysteine at a position corresponding to 112), canine apoE (arginine at the corresponding site), and cysteamine-treated rabbit apoE (cysteine converted to a positively charged residue). Surprisingly, all distributed like human apoE3, suggesting that positive charge at a position corresponding to 112 was not directly responsible for the isoform preference and that other residues in the amino-terminal domain were involved. To determine which residues were involved, the structure of the apoE4 22-kDa fragment (the amino-terminal two-thirds of the molecule) was determined to 2.5 A by x-ray crystallography. Compared with the known four-helix bundle structure of apoE3, the only significant differences in the apoE4 structure were that glutamic acid 109 formed a salt bridge with arginine 112 and that the arginine 61 side chain was displaced to a new position. Site-directed mutagenesis of glutamic acid 109 in apoE3 and arginine 61 in apoE4 demonstrated that the position of the arginine 61 side chain in apoE4 was critical in determining apoE4 lipoprotein distribution, suggesting that arginine 61 interacted with the carboxyl-terminal domain to direct binding to VLDL.

Salt bridge relay triggers defective LDL receptor binding by a mutant apolipoprotein.

BACKGROUND: Apolipoprotein-E (apo-E), a 34kDa blood plasma protein, plays a key role in directing cholesterol transport via its interaction with the low density lipoprotein (LDL) receptor. The amino-terminal domain of apo-E forms an unusually elongated four-helix bundle arranged such that key basic residues involved in LDL receptor binding form a cluster at the end of one of the helices. A common apo-E variant, apo-E2, corresponding to the single-site substitution Arg158-->Cys, displays minimal LDL receptor binding and is associated with significant changes in plasma cholesterol levels and increased risk of coronary heart disease. Surprisingly, the site of mutation in this variant is physically well removed (> 12A) from the cluster of LDL receptor binding residues. RESULTS: We now report the refined crystal structure of the amino-terminal domain of apo-E2, at a nominal resolution of 3.0A. This structure reveals significant conformational changes relative to the wild-type protein that may account for reduced LDL receptor binding. Removal of the Arg158 side chain directly disrupts a pair of salt bridges, causing a compensatory reorganization of salt bridge partners that dramatically alters the charge surface presented by apo-E to its receptor. CONCLUSIONS: It is proposed that the observed reorganization of surface salt bridges is responsible for the decreased receptor binding by apo-E2. This reorganization, essentially functioning as a mutationally induced electrostatic switch to turn off receptor binding, represents a novel mechanism for the propagation of conformational changes over significant distances.

Biological implications of a 3 A structure of dimeric antithrombin.

BACKGROUND: Antithrombin, a member of the serpin family of inhibitors, controls coagulation in human plasma by forming complexes with thrombin and other coagulation proteases in a process greatly accelerated by heparin. The structures of several serpins have been determined but not in their active conformations. We have determined the structure of intact antithrombin in order to study its mechanism of activation, particularly with respect to heparin, and the dysfunctions of this mechanism that predispose individuals to thrombotic disease. RESULTS: The crystal structure of a dimer of one active and one inactive molecule of antithrombin has been determined at 3 A. The first molecule has its reactive-centre loop in a predicted active conformation compatible with initial entry of two residues into the main beta-sheet of the molecule. The inactive molecule has a totally incorporated loop as in latent plasminogen activator inhibitor-1. The two molecules are linked by the reactive loop of the active molecule which has replaced a strand from another beta-sheet in the latent molecule. CONCLUSION: The structure, together with identified mutations affecting its heparin affinity, allows the placement of the heparin-binding site on the molecule. The conformation of the two forms of antithrombin demonstrates the extraordinary mobility of the reactive loop in the serpins and provides insights into the folding of the loop required for inhibitory activity together with the potential modification of this by heparin. The mechanism of dimerization is relevant to the polymerization that is observed in diseases associated with variant serpins.

Crystallization and preliminary X-ray diffraction analysis of two conformations of intact human antithrombin.

Human antithrombin has been crystallized by microdialysis at pH 6.7 using 18% (w/v) polyethylene glycol-4000 as precipitant. Under these conditions two crystal forms grew. The first started growing after ten days, diffracted to 3.0 A resolution and belongs to the monoclinic space group P2(1) with two molecules in the asymmetric unit and unit cell dimensions a = 70.1 A, b = 101.5 A, c = 90.5 A and beta = 105.9 A. The other crystal form took more than three months to appear, diffracted to 5.5 A and belongs to the hexagonal space group of either P6(1) or P6(5) with unit cell dimensions of a = b = 99.3 A and c = 152.9 A and two molecules in the asymmetric unit. The antithrombin redissolved from the monoclinic crystals was shown both by SDS-polyacrylamide gel electrophoresis and by protein sequence analysis to be intact while that from the hexagonal crystals was cleaved in the reactive centre loop between the P'2 and P'3 (i.e. Leu-Asn) residues. Further analysis of the intact inhibitor from the monoclinic crystals indicated that the antithrombin was present in two different conformations; an active form which could inhibit thrombin and form a stable complex with the protease, and a form which was inactive as an inhibitor and which also did not act as a substrate for thrombin. This latter form also had a low affinity for heparin and in these ways resembles latent antithrombin. The active material from the monoclinic crystals had an association rate constant with thrombin in the presence of heparin (kass) of 7.5 x 10(7) M-1 s-1 (kass for native antithrombin = 8.2 (+/- 1.0) x 10(7) M-1 s-1) indicating it still had effective heparin cofactor activity. X-ray diffraction analysis also suggests that two different protein conformations exist within the monoclinic crystals. Whereas the rotation function peak heights are equal for both molecules in the asymmetric unit using the structure of intact ovalbumin as a search model, one of the two molecules gives a much clearer signal than the other when the structures of the two cleaved serpins, alpha 1-antitrypsin and alpha 1-antichymotrypsin are used.

Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation.

Apolipoprotein (apo) E and the two B apolipoproteins, apoB48 and apoB100, are important proteins in human lipoprotein metabolism. Commonly occurring polymorphisms in the genes for apoE and apoB result in amino acid substitutions that produce readily detectable phenotypic differences in these proteins. We studied changes in apoE and apoB phenotypes before and after liver transplantation to gain new insights into apolipoprotein physiology. In all 29 patients that we studied, the postoperative serum apoE phenotype of the recipient, as assessed by isoelectric focusing, converted virtually completely to that of the donor, providing evidence that greater than 90% of the apoE in the plasma is synthesized by the liver. In contrast, the cerebrospinal fluid apoE phenotype did not change to the donor's phenotype after liver transplantation, indicating that most of the apoE in CSF cannot be derived from the plasma pool and therefore must be synthesized locally. The apoB100 phenotype (assessed with immunoassays using monoclonal antibody MB19, an antibody that detects a two-allele polymorphism in apoB) invariably converted to the phenotype of the donor. In four normolipidemic patients, we determined the MB19 phenotype of both the apoB100 and apoB48 in the "chylomicron fraction" isolated from plasma 3 h after a fat-rich meal. Interestingly, the apoB100 in the chylomicron fraction invariably had the phenotype of the donor, indicating that the vast majority of the large, triglyceride-rich apoB100-containing lipoproteins that appear in the plasma after a fat-rich meal are actually VLDL of hepatic origin. The MB19 phenotype of the apoB48 in the plasma chylomicron fraction did not change after liver transplantation, indicating that almost all of the apoB48 in plasma chylomicrons is derived from the intestine. These results were consistent with our immunocytochemical studies on intestinal biopsy specimens of organ donors; using apoB-specific monoclonal antibodies, we found evidence for apoB48, but not apoB100, in donor intestinal biopsy specimens.