Crystal structure of PHYHD1A, a 2OG oxygenase related to phytanoyl-CoA hydroxylase

Phytanoyl-CoA hydroxylase (PAHX) catalyzes an important step in the metabolism of the fatty acid side chain of chlorophyll. PHYHD1 exists in three isoforms and is the closest human homologue of PAHX. We show that like PAHX, the PHYHD1A but likely not the PHYHD1B/C isoforms, is a functional Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase. Crystallographic and biochemical analyses reveal that PHYHD1A has the double-stranded β-helix fold and Fe(II) and cosubstrate binding residues characteristic of the 2-oxoglutarate dependent oxygenases and catalyzes the conversion of 2-oxoglutarate to succinate and CO₂ in an iron-dependent manner. However, PHYHD1A did not couple 2OG turnover to the hydroxylation of acyl-coenzyme A derivatives that are substrates for PAHX, implying that it is not directly involved in phytanoyl coenzyme-A metabolism.     

Structure of human phytanoyl-CoA 2-hydroxylase identifies molecular mechanisms of Refsum disease

Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the initial alpha-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG oxygenases, PAHX possesses a double-stranded beta-helix core, which supports three iron binding ligands (His(175), Asp(177), and His(264)); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys(191)) 6.7 A from the Fe(II) and two further histidine residues (His(155) and His(281)) at its active site distinguishes it from that of the other human 2OG oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro(173), His(175), Gln(176), Asp(177), and His(220)) and 2OG binding sites (Trp(193), Glu(197), Ile(199), Gly(204), Asn(269), and Arg(275)). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG oxygenases.     

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.     

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site

XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.     

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.     

Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH)

A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.     

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.     

Chemical modification of spinach plastocyanin using 4-chloro-3,5-dinitrobenzoic acid: characterization of four singly-modified forms

Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.     

N-terminus of the photosystem II manganese stabilizing protein: effects of sequence elongation and truncation

The importance of the N-terminal domain of manganese stabilizing protein in binding to photosystem II has been previously demonstrated [Eaton-Rye and Murata (1989) Biochim. Biophys. Acta 977, 219-226; Odom and Bricker (1992) Biochemistry 31, 5616-5620]. In this paper, we report results from a systematic study of functional and structural consequences of N-terminal elongation and truncation of manganese stabilizing protein. Precursor manganese stabilizing protein is the unprocessed wild-type protein, which carries an N-terminal extension of 84 amino acids in the form of its chloroplastic signal peptide. Despite its increased size, this protein is able to reconstitute O(2) evolution activity to levels observed with the mature, processed protein, but it also binds nonspecifically to PSII. Truncation of wild-type manganese stabilizing protein by site-directed mutagenesis to remove three N-terminal amino acids, resulting in a mutant called DeltaG3M, causes no loss of activity reconstitution, but this protein also exhibits nonspecific binding. Further truncation of the wild-type protein by ten N-terminal amino acids, producing DeltaE10M, limits binding of manganese stabilizing protein to 1 mol/mol of photosystem II and decreases activity reconstitution to about 65% of that obtained with the wild-type protein. Because two copies of wild type normally bind to photosystem II, amino acids in the domain (4)K-(10)E must be involved in the binding of one copy of manganese stabilizing protein to photosystem II. Spectroscopic analysis (CD and UV spectra) reveals that N-terminal elongation and deletion of manganese stabilizing protein influence its overall conformation, even though secondary structure content is not perturbed. Our data suggest that the solution structure of manganese stabilizing protein attains a more compact solution structure upon removal of N-terminal amino acids.     

Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (g(II) = 2.232; A(II) = 4.4 mT) and type 2 Cu centers (g(II) = 2.304; A(II) = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 microM NO(2)(-) x min(-1) x mg(-1).     

Copper-induced structural propensities of the amyloidogenic region of human prion protein

Transmissible spongiform encephalopathies are associated with the misfolding of the cellular Prion Protein (PrP^C) to an abnormal protein isoform, called scrapie prion protein (PrP^Sc). The structural rearrangement of the fragment of N-terminal domain of the protein spanning residues 91–127 is critical for the observed structural transition. The amyloidogenic domain of the protein encloses two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for metal ion binding. Previous studies have shown that Cu(II) sequestration by both sites may modulate the peptide’s tendency to aggregation as it inflicts the hairpin-like structure that stabilizes the transition states leading to β-sheet formation. On the other hand, since both His sites differ in their ability to Cu(II) sequestration, with His-111 as a preferred binding site, we found it interesting to test the role of Cu(II) coordination to this single site on the structural properties of amyloidogenic domain. The obtained results reveal that copper binding to His-111 site imposes precise backbone bending and weakens the natural tendency of apo peptide to β-sheet formation.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase. Identification of residues involved in metallocenter formation and substrate binding

2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. The active site structures of a small number of enzymes within the alpha-KG-dependent dioxygenase superfamily have been characterized and shown to have a similar HXDX(50-70)HX(10)RXS arrangement of residues that make up the binding sites for Fe(II) and alpha-KG. TfdA does not have obvious homology to the dioxygenases containing the above motif but is related in sequence to eight other enzymes in the superfamily that form a distinct consensus sequence (HX(D/E)X(138-207) HX(10)R/K). Variants of TfdA were created to examine the roles of putative metal-binding residues and the functions of the other seven histidines in this protein. The H167A, H200A, H213A, H245A, and H262A forms of TfdA formed inclusion bodies when overproduced in Escherichia coli DH5alpha; however, these proteins were soluble when fused to the maltose-binding protein (MBP). MBP-TfdA exhibited kinetic parameters similar to the native enzyme. The H8A and H235A variants were catalytically similar to wild-type TfdA. MBP-H213A and H216A TfdA have elevated K(m) values for 2,4-D, and the former showed a decreased k(cat), suggesting these residues may affect substrate binding or catalysis. The H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A variants of TfdA were inactive. Gel filtration analysis revealed that the latter two proteins were highly aggregated. The remaining four inactive variants were examined in their Cu(II)-substituted forms by EPR and electron spin-echo envelope modulation (ESEEM) spectroscopic methods. Changes in EPR spectra upon addition of substrates indicated that copper was present at the active site in the H113A and D115A variants. ESEEM analysis revealed that two histidines are bound equatorially to the copper in the D115A and MBP-H167A TfdA variants. The experimental data and sequence analysis lead us to conclude that His-113, Asp-115, and His-262 are likely metal ligands in TfdA and that His-213 may aid in catalysis or binding of 2,4-D.     

Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A.

Phytanoyl-CoA 2-hydroxylase (PAHX), an iron(II) and 2-oxoglutarate-dependent oxygenase, catalyses an essential step in the mammalian metabolism of beta-methylated fatty acids. Phytanoyl-CoA was synthesised and used to develop in vitro assays for PAHX. The product of the reaction was confirmed as 2-hydroxyphytanoyl-CoA by NMR and mass spectrometric analyses. In accord with in vivo analyses, hydroxylation of both 3R and 3S epimers of the substrate was catalysed by PAHX. Both pro- and mature- forms of PAHX were fully active.     

Roles of phytanoyl-CoA alpha-hydroxylase in mediating the expression of human coagulation factor VIII

The coagulation factor VIII (FVIII) is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Previous transfection studies demonstrated that factor VIII was 10-100-fold less efficiently expressed than the homologous coagulation factor, factor V. To investigate the regulatory mechanisms of FVIII synthesis and secretion, we used the yeast two-hybrid system as an approach to search for proteins that associated with FVIII. The A2 domain (337-740 amino acids) of factor VIII (FVIII-A2) was used as a bait and phytanoyl-CoA alpha-hydroxylase (PAHX) was identified as a binding protein of FVIII-A2. PAHX had potential to interact with the residues 373-508 within the A2 domain, but not with A1 and A3 (the homologous domains of A2). The interaction between the A2 domain and PAHX was independent of the type 2 peroxisomal targeting signal (PTS2) of PAHX. Overexpression of PAHX in FVIII-produced cells decreased the expression of FVIII by about 70%. The elevated expression of von Willebrand factor had no effect on the suppression of FVIII secretion by PAHX. Expression of the green fluorescent PAHX fusion protein in SMMC-7721 cells affected the intracellular trafficking of FVIII-A2. These results suggested that the interaction between PAHX and FVIII-A2 was in part responsible for the low-level expression of factor VIII.     

Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.     

Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins

Heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2alpha, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory Cys-Pro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation

Islet amyloid deposits are a characteristic pathological hallmark of type 2 diabetes mellitus. Islet amyloid polypeptide (IAPP), also referred to as amylin, aggregates in the islet extracellular space to form amyloid deposits in up to 95% of patients with the disease. IAPP is stored with insulin in beta-islet cells and is processed in parallel by subtilisin-like prohormone convertases prior to secretion. There is indirect evidence that normal processing of the prohormone precursor, proIAPP, at the N-terminal cleavage site is defective in type 2 diabetes and results in secretion of an N-terminal extended proIAPP intermediate. The N-terminal flanking region of proIAPP is detected in amyloid deposits; however, the C-terminal flanking region is not. Immunohistochemical studies implicate the presence of the heparan sulfate proteoglycan (HSPG) perlecan in islet amyloid deposits, suggesting a role for HSPGs in mediating amyloid deposition in type 2 diabetes and implicating a binding domain in the N-terminus of proIAPP. Initial studies of proIAPP indicated that the HSPG binding region is contained within the first 30 residues. Here, we characterize the potential HSPG binding site of proIAPP in detail by analyzing a set of peptide fragments. Binding is tighter at low pH due to protonation of histidine residues. Deletion studies show that Arg-22 and His-29 play a role in binding. Reduction of the Cys-13 to Cys-18 disulfide leads to a noticeable decrease in binding. We demonstrate the ability of heparan sulfate to induce amyloid formation in N-terminal fragments of proIAPP. The oxidized peptide forms amyloid more rapidly than the reduced variant in the presence of heparan sulfate, but the reduced peptide ultimately forms more extensive amyloid deposits. The potential implications for islet amyloid formation in vivo are discussed.