Complexation of Cm(III) with the recombinant N-lobe of human serum transferrin studied by time-resolved laser fluorescence spectroscopy (TRLFS).

The complexation of Cm(III) with the recombinant N-lobe of human serum transferrin (hTf/2N) is investigated in the pH range from 4.0 to 11.0 using TRLFS. At pH ≥ 7.4 a Cm(III) hTf/2N species is formed with Cm(III) bound at the Fe(III) binding site. The results are compared with Cm(III) transferrin interaction at the C-lobe and indicate the similarity of the coordination environment of the C- and N-terminal binding sites with four amino acid residues of the protein, two H2O molecules and three additional ligands (e.g. synergistic anions such as carbonate) in the first coordination sphere. Measurements at c(carbonate)tot = 0.23 mM (ambient carbonate concentration) and c(carbonate)tot = 25 mM (physiological carbonate concentration) show that an increase of the total carbonate concentration suppresses the formation of the Cm(III) hTf/2N species significantly. Additionally, the three Cm(III) carbonate species Cm(CO3)(+), Cm(CO3)2(-) and Cm(CO3)3(3-) are formed successively with increasing pH. In general, carbonate complexation is a competing reaction for both Cm(III) complexation with transferrin and hTf/2N but the effect is significantly higher for the half molecule. At c(carbonate)tot = 0.23 mM the complexation of Cm(III) with transferrin and hTf/2N starts at pH ≥ 7.4. At physiological carbonate concentration the Cm(III) transferrin species II forms at pH ≥ 7.0 whereas the Cm(III) hTf/2N species is not formed until pH > 10.0. Hence, our results reveal significant differences in the complexation behavior of the C-terminal site of transferrin and the recombinant N-lobe (hTf/2N) towards trivalent actinides.

Severe prothrombin deficiency caused by prothrombin-Edmonton (R-4Q) combined with a previously undetected deletion.

BACKGROUND: During infancy, a male patient experienced several life-threatening bleeding episodes. Standard coagulation tests revealed that the patient's plasma prothrombin activity was 8%, while his father's and mother's levels were 74% and 62%, respectively. OBJECTIVES: A molecular genetic approach was used to determine the molecular basis of prothrombin deficiency within the family. PATIENT/METHODS: Prothrombin genomic DNA fragments were amplified by using the polymerase chain reaction (PCR). In addition, liver cDNA fragments were amplified from the patient by using reverse transcription (RT) and PCR. The nucleotide sequences of the DNA fragments were determined. RESULTS: A novel, heterozygous point mutation (g.1755 G > A, named prothrombin-Edmonton) was detected in the patient and his mother, resulting in the mutation of Arg-4 in the prothrombin propeptide to Gln (R-4Q). RT-PCR analysis of the patient's liver sample demonstrated the presence of two mRNA transcripts that differed by the presence or absence of exon 11. Real-time PCR analysis on genomic DNA and cDNA confirmed a deletion (g.10435_10809del) in the paternal allele. CONCLUSIONS: The patient has a maternally-inherited point mutation (R-4Q) and a paternally-inherited deletion. By analogy with the previously reported factor IX San Dimas, the R-4Q mutation probably causes under-carboxylation of prothrombin and poor cleavage of the propeptide in the hepatocyte. The deletion probably results in a polypeptide that lacks 50 amino acids from the protease domain; this is likely to impair folding, secretion, stability and/or activity of the truncated prothrombin. The two mutations combine to give the prothrombin deficiency observed in the patient.

Determinants of specificity in coagulation proteases.

Proteases play diverse roles in a variety of essential biological processes, both as non-specific catalysts of protein degradation and as highly specific agents that control physiologic events. Here, we review the mechanisms of substrate specificity employed by serine proteases and focus our discussion on coagulation proteases. We dissect the interplay between active site and exosite specificity and how substrate recognition is regulated allosterically by Na+ binding. We also draw attention to a functional polarity that exists in the serine protease fold, which sheds light on the structural linkages between the active site and exosites.

Expression, purification, and characterization of recombinant nonglycosylated human serum transferrin containing a C-terminal hexahistidine tag.

Attachment of a hexa-His tag is a common strategy in recombinant protein production. The use of such a tag greatly simplifies the purification of the protein from the complex mixture of other proteins in the media or cell extract. We describe the production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at their carboxyl-terminal ends. One of the constructs comprises the entire coding region for hTF (residues 1-679), while the other lacks the final three carboxyl-terminal amino acids. After insertion of the His-tagged hTFs into the pNUT vector, transfection into baby hamster kidney (BHK) cells, and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium. Average maximum expression levels of the His-tagged hTFs were about 40 mg/L compared to an average maximum of 50 mg/L for hTF-NG. The first step of purification involved an anion exchange column. The second step utilized a Poros metal chelate column preloaded with copper from which the His-tagged sample was eluted with a linear imidazole gradient. The His-tagged hTFs were characterized and compared to both recombinant hTF-NG and glycosylated hTF from human serum. The identity of each of the His-tagged hTFs constructs was verified by electrospray mass spectroscopy. In summary, the His-tagged hTF constructs simplify the purification of these metal-binding proteins with minimal effects on many of their physical properties. The His-tagged hTFs share many features common to hTF, including reversible iron binding, reactivity with a monoclonal antibody, and presence as a monomer in solution.

Ligand variation in the transferrin family: the crystal structure of the H249Q mutant of the human transferrin N-lobe as a model for iron binding in insect transferrins.

Proteins of the transferrin (Tf) family play a central role in iron homeostasis in vertebrates. In vertebrate Tfs, the four iron-binding ligands, 1 Asp, 2 Tyr, and 1 His, are invariant in both lobes of these bilobal proteins. In contrast, there are striking variations in the Tfs that have been characterized from insect species; in three of them, sequence changes in the C-lobe binding site render it nonfunctional, and in all of them the His ligand in the N-lobe site is changed to Gln. Surprisingly, mutagenesis of the histidine ligand, His249, to glutamine in the N-lobe half-molecule of human Tf (hTf/2N) shows that iron binding is destabilized and suggests that Gln249 does not bind to iron. We have determined the crystal structure of the H249Q mutant of hTf/2N and refined it at 1.85 A resolution (R = 0.221, R(free) = 0.246). The structure reveals that Gln249 does coordinate to iron, albeit with a lengthened Fe-Oepsilon1 bond of 2.34 A. In every other respect, the protein structure is unchanged from wild-type. Examination of insect Tf sequences shows that the K206.K296 dilysine pair, which aids iron release from the N-lobes of vertebrate Tfs, is not present in the insect proteins. We conclude that substitution of Gln for His does destabilize iron binding, but in the insect Tfs this is compensated by the loss of the dilysine interaction. The combination of a His ligand with the dilysine pair in vertebrate Tfs may have been a later evolutionary development that gives more sophisticated pH-mediated control of iron release from the N-lobe of transferrins.

Crystal structures and iron release properties of mutants (K206A and K296A) that abolish the dilysine interaction in the N-lobe of human transferrin.

Human transferrin (Tf) is responsible for the binding and transport of iron in the bloodstream of vertebrates. Delivery of this bound iron to cells occurs by a process of receptor-mediated endocytosis during which Tf releases its iron at the reduced endosomal pH of approximately 5.6. Iron release from Tf involves a large conformational change in which the two domains that enclose the binding site in each lobe move apart. We have examined the role of two lysines, Lys206 and Lys296, that form a hydrogen-bonded pair close to the N-lobe binding site of human Tf and have been proposed to form a pH-sensitive trigger for iron release. We report high-resolution crystal structures for the K206A and K296A mutants of the N-lobe half-molecule of Tf, hTf/2N, and quantitative iron release data on these mutants and the double mutant K206A/K296A. The refined crystal structures (for K206A, R = 19.6% and R(free) = 23.7%; for K296A, R= 21.2% and R(free) = 29.5%) reveal a highly conserved hydrogen bonding network in the dilysine pair region that appears to be maintained even when individual hydrogen bonding groups change. The iron release data show that the mutants retain iron to a pH 1 unit lower than the pH limit of wild type hTf/2N, and release iron much more slowly as a result of the loss of the dilysine interaction. Added chloride ions are shown to accelerate iron release close to the pH at which iron is naturally lost and the closed structure becomes destabilized, and to retard it at higher pH.

Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe.

Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp(8), Trp(128) and Trp(264), located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV-visible, fluorescence and NMR spectra of hTF/2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp(8) resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two beta-sheets containing Trp(8) and Lys(296) respectively. The fluorescence of Trp(8) may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp(8)-->Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence of Trp(128) results from the nearby His(119) residue. Difference-fluorescence spectra reveal that any protein containing Trp(128) shows a blue shift upon binding metal ion, and the NMR signal of Trp(128) broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp(128) is a major fluorescent and NMR reporter group for metal binding, and possibly for cleft closure in hTF/2N. Trp(264) is located on the surface of the protein and does not connect to any functional residues. This explains the facts that Trp(264) is the major contributor to both the absorbance and fluorescence spectra, has a strong NMR signal and the mutation at Trp(264) has little effect on the iron-binding and release behaviours of the protein.

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein.

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.

The chloride effect is related to anion binding in determining the rate of iron release from the human transferrin N-lobe.

The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, 207, and 215 of thrombin.

Conservative Trp-to-Phe mutations were individually created in human thrombin at positions 60d, 96, 148, 207, and 215. Fluorescence intensities for these residues varied by a factor of 6. Residues 60d, 96, 148, and 215 transferred energy to the thrombin inhibitor 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5- pentanediyl)amide efficiently, but residue 207 did not. Intensities correlated inversely with exposure to solvent, and measured and theoretical energy transfer efficiencies agreed well. Function was measured with respect to fibrinogen clotting, platelet and factor V activation, inhibition by antithrombin, and the thrombomodulin-dependent activation of protein C and thrombin-activable fibrinolysis inhibitor (TAFI). All activities of W96F and W207F ranged from 74 to 154% of the wild-type activity. This was also true for W148F, except for inhibition by antithrombin, where it showed 60% activity. W60dF was deficient by 30, 57, and 43% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1006)), respectively. W215F was deficient by 90, 55, and 56% with fibrinogen clotting, platelet activation, and factor V cleavage (Arg(1536)). With protein C and TAFI, W96F, W148F, and W207F were normal. W60dF, however, was 76 and 23% of normal levels with protein C and TAFI, respectively. In contrast, W215F was 25 and 124% of normal levels in these reactions. Thus, many activities of thrombin are retained upon substitution of Trp with Phe at positions 96, 148, and 207. Trp(60d), however, appears to be very important for TAFI activation, and Trp(215) appears to very important for clotting and protein C activation.

Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron.

The X-ray crystallographic structures of two mutants (K206Q and H207E) of the N-lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 A, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H-bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe.

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release.

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.

[13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.

The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, (13)C NMR of the [epsilon-(13)C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met(26) and Met(109), has been corrected. A series of NMR spectra for the complexes of (13)C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met(109)-->Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized.

Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence.

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.

Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris.

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.