Iron release from transferrin, its C-lobe, and their complexes with transferrin receptor: presence of N-lobe accelerates release from C-lobe at endosomal pH

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. Although the two lobes exhibit 45% sequence identity and identical ligand structures of their iron-binding sites (one in each lobe), they differ in their iron-binding properties and their responsiveness to complex formation with the transferrin receptor. A variety of interlobe interactions modulating these iron-binding functions has been described. We have now studied the kinetics of iron release to pyrophosphate from the isolated recombinant C-lobe and from that lobe in the intact protein, each free and bound to receptor. The striking finding is that the rates of iron release at the pH of the endosome to which transferrin is internalized by the iron-dependent cell are similar in the free proteins but 18 times faster from full-length monoferric transferrin selectively loaded with iron in the C-lobe than from isolated C-lobe when each is complexed to the receptor. The possibility that the faster release in the receptor complex of the full-length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.     

Structural allostery and binding of the transferrin*receptor complex

The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.     

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) – haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb – Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.     

Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin

The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.     

Mutational analysis of C-lobe ligands of human serum transferrin: insights into the mechanism of iron release

Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive; each monoferric hTF has a free energy of binding that is 82% of diferric hTF.     

Lysozyme modification by the fenton reaction and gamma radiation

A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(II)/H2 O2) or hydroxyl radicals produced by y radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. Gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr > Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.     

Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor.

The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution

Distal side tryptophan and tyrosine have been shown to be essential in the catalase but not the peroxidase activity of bifunctional catalase-peroxidases (KatGs). Recently published crystal structures suggest that both residues could be part of a novel adduct including in addition a conserved methionine. A mass spectrometric analysis of the tryptic peptides from recombinant wild-type Synechocystis KatG and the variants Trp122Phe, Tyr249Phe and Met275Ile confirms that this novel adduct really exists in solution and thus may be common to all KatGs. Exchange of either Trp122 or Tyr249 prevents cross-linking, whereas exchange of Met275 still allowed bond formation between Trp122 and Tyr249. It is proposed that the covalent bond between Trp and Tyr may form before that between Tyr and Met. The findings are discussed with respect to the mechanism of cross-linking and its role in KatG catalysis.     

A new method for obtaining human transferrin C-lobe in the native conformation: preparation and properties

Eukaryotic transferrins comprise a class of bilobal iron-binding proteins in which each lobe carries a single binding site. Although expression of full-length transferrins and their N-terminal lobes, in wild-type and mutated forms, has been successfully accomplished by several laboratories, expression of C-lobes has been much less satisfactory. A possible explanation of the difficulty is that proper folding of the C-lobe, with its 11 disulfide bonds, depends on prior synthesis and proper folding of the N-lobe. We have therefore developed a new strategy, introducing a specific factor Xa cleavage site in the interlobe-connecting strand to permit separation of the lobes after expression of the full-length protein. The resulting protein was expressed in satisfactory yield, >20 mg/L, and could be easily and completely cleaved to yield two distinguishable fragments representing N- and C-lobes, respectively. Retaining the glycosylation sites, found only in the C-lobe, made it possible to separate the fragments from each other by ConA affinity chromatography. The isolated C-lobe so obtained displayed spectroscopic and kinetic features of the C-lobe in native transferrin and was competent as an iron donor for K562 cells to which it bound in saturable fashion inhibitable by native diferric transferrin. Since the N-lobe by itself will neither bind nor donate iron to cells, the primary receptor-recognition site of transferrin resides in its C-lobe.     

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.     

Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release

Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.     

Fluorescence properties of glutamine-binding protein from Escherichia coli and its complex with glutamine

In this work, the fluorescence of glutamine-binding protein (GlnBP) and its complex with glutamine (GlnBP/Gln) in native and unfolded forms was studied. The experimental data were interpreted on the basis of the results of the analysis of Trp and Tyr microenvironments taking into the account the data for GlnBP mutated forms Trp32Phe(Tyr) and Trp220Phe(Tyr), which have been obtained by Axelsen et al. (Biophys. J. 1991, 60, 650-659). This allowed us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of the fluorescence characteristics of GlnBP and GlnBP/Gln, and the uncommon effect of the excess of the fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm respect to the excitation at 280 nm. The last effect is explained by the spectral dependence of the Trp 32 and Trp 220 contributions to the protein absorption. The protein Trp fluorescence dependence on the excitation wavelength must be taken into account for the evaluation of the Tyr residues contribution to the bulk fluorescence of protein, and in principle, it also may be used for the development of an approach for the decomposition of a multicomponent protein fluorescence spectrum.     

Roles of the P1, P2, and P3 residues in determining inhibitory specificity of kallistatin toward human tissue kallikrein

Kallistatin is a serpin with a unique P1 Phe, which confers an excellent inhibitory specificity toward tissue kallikrein. In this study, we investigated the P3-P2-P1 residues (residues 386-388) of human kallistatin in determining inhibitory specificity toward human tissue kallikrein by site-directed mutagenesis and molecular modeling. Human kallistatin mutants with 19 different amino acid substitutions at each P1, P2, or P3 residue were created and purified to compare their kallikrein binding activity. Complex formation assay showed that P1 Arg, P1 Phe (wild type), P1 Lys, P1 Tyr, P1 Met, and P1 Leu display significant binding activity with tissue kallikrein among the P1 variants. Kinetic analysis showed the inhibitory activities of the P1 mutants toward tissue kallikrein in the order of P1 Arg > P1 Phe > P1 Lys >/= P1 Tyr > P1 Leu >/= P1 Met. P1 Phe displays a better selectivity for human tissue kallikrein than P1 Arg, since P1 Arg also inhibits several other serine proteinases. Heparin distinguishes the inhibitory specificity of kallistatin toward kallikrein versus chymotrypsin. For the P2 and P3 variants, the mutants with hydrophobic and bulky amino acids at P2 and basic amino acids at P3 display better binding activity with tissue kallikrein. The inhibitory activities of these mutants toward tissue kallikrein are in the order of P2 Phe (wild type) > P2 Leu > P2 Trp > P2 Met and P3 Arg > P3 Lys (wild type). Molecular modeling of the reactive center loop of kallistatin bound to the reactive crevice of tissue kallikrein indicated that the P2 residue required a long and bulky hydrophobic side chain to reach and fill the hydrophobic S2 cleft generated by Tyr(99) and Trp(219) of tissue kallikrein. Basic amino acids at P3 could stabilize complex formation by forming electrostatic interaction with Asp(98J) and hydrogen bond with Gln(174) of tissue kallikrein. Our results indicate that tissue kallikrein is a specific target proteinase for kallistatin.     

Composition of pH-sensitive triad in C-lobe of human serum transferrin. Comparison to sequences of ovotransferrin and lactoferrin provides insight into functional differences in iron release

The transferrins (TF) are a family of bilobal glycoproteins that tightly bind ferric iron. Each of the homologous N- and C-lobes contains a single iron-binding site situated in a deep cleft. Human serum transferrin (hTF) serves as the iron transport protein in the blood; circulating transferrin binds to receptors on the cell surface, and the complex is internalized by endocytosis. Within the cell, a reduction in pH leads to iron release from hTF in a receptor-dependent process resulting in a large conformational change in each lobe. In the hTF N-lobe, two critical lysines facilitate this pH-dependent conformational change allowing entry of a chelator to capture the iron. In the C-lobe, the lysine pair is replaced by a triad of residues: Lys534, Arg632, and Asp634. Previous studies show that mutation of any of these triad residues to alanine results in significant retardation of iron release at both pH 7.4 and pH 5.6. In the present work, the role of the three residues is probed further by conversion to the residues observed at the equivalent positions in ovotransferrin (Q-K-L) and human lactoferrin (K-N-N) as well as a triad with an interchanged lysine and arginine (K534R/R632K). As expected, all of the constructs bind iron and associate with the receptor with nearly the same K(D) as the wild-type monoferric hTF control. However, interesting differences in the effect of the substitutions on the iron release rate in the presence and absence of the receptor at pH 5.6 are observed. Additionally, titration with KCl indicates that position 632 must have a positively charged residue to elicit a robust rate acceleration as a function of increasing salt. On the basis of these observations, a model for iron release from the hTF C-lobe is proposed. These studies provide insight into the importance of charge and geometry of the amino acids at these positions as a partial explanation for differences in behavior of individual TF family members, human serum transferrin, ovotransferrin, and lactoferrin. The studies collectively highlight important features common to both the N- and C-lobes of TF and the critical role of the receptor in iron release.     

Single-site mutations on the catalase–peroxidase from Sinorhizobium meliloti: role of the distal Gly and the three amino acids of the putative intrinsic cofactor

KatB is the only catalase–peroxidase identified so far in Sinorhizobium meliloti. It plays a housekeeping role, as it is expressed throughout all the growth phases of the free-living bacterium and also during symbiosis. This paper describes the functional and structural characterization of the KatB mutants Gly303Ser, Trp95Ala, Trp95Phe, Tyr217Leu, Tyr217Phe and Met243Val carried out by optical and electron spin resonance spectroscopy. The aim of this work was to investigate the involvement of these residues in the catalatic and/or peroxidatic reaction and falls in the frame of the open dispute around the factors that influence the balance between catalatic and peroxidatic activity in heme enzymes. The Gly303 residue is not conserved in any other protein of this family, whereas the Trp95, Tyr217 and Met243 residues are thought to form an intrinsic cofactor that is likely to play a role in intramolecular electron transfer. Spectroscopic investigations show that the Gly303Ser mutant is almost similar to the wild-type KatB and should not be involved in substrate binding. Mutations on Trp95, Tyr217 and Met243 clear out the catalatic activity completely, whereas the peroxidatic activity is maintained or even increased with respect to that of the wild-type enzyme. The k _cat values obtained for these mutants suggest that Trp95 and Tyr217 form a huge delocalized system that provides a pathway for electron transfer to the heme. Conversely, Met243 is likely to be placed close to the binding site of the organic molecules and plays a crucial role in substrate docking.     

The intrinsic fluorescence of the recombinant human leukocyte interferon-alpha A and fibroblast interferon-beta 1

The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).