High-resolution X-ray structures of human apoferritin H-chain mutants correlated with their activity and metal-binding sites

Ferritins are a family of proteins distributed widely in nature. In bacterial, plant, and animal cells, ferritin appears to serve as a soluble, bioavailable, and non-toxic form of iron provider. Ferritins from animal sources are heteropolymers composed of two types of subunit, H and L, which differ mainly by the presence (H) or absence (L) of active ferroxidase centres. We report the crystallographic structures of four human H apoferritin variants at a resolution of up to 1.5 Angstrom. Crystal derivatives using Zn(II) as redox-stable alternative for Fe(II), allows us to characterize the different metal-binding sites. The ferroxidase centre, which is composed of sites A and B, binds metal with a preference for the A site. In addition, distinct Zn(II)-binding sites were found in the 3-fold axes, 4-fold axes and on the cavity surface near the ferroxidase centre. To study the importance of the distance of the two metal atoms in the ferroxidase centre, single and double replacement of glutamate 27 (site A) and glutamate 107 (site B), the two axial ligands, by aspartate residues have been carried out. The consequences for metal binding and the correlation with Fe(II) oxidation rates are discussed.     

Spin-resolved X-ray absorption near edge structure (XANES) simulation of metmyoglobin

Spin resolved multiple scattering (MS) calculations of Fe K-edge X-ray Absorption Near Edge Structure (XANES) of myoglobin are reported for the first time. The observed differences of the Fe K-edge XANES spectra of sperm whale metmyoglobin under spin transition as a function of temperature have been studied. The method allows one to compute separately spin effects and local structural effects. The results show that spin effects are confined in the absorption rising edge in the range 7111–7130 eV, while purely structural effects are dominant in the range 7130–7170 eV. Symmetry changes of the Fe coordination sphere mainly related to its movement towards the heme plane, coupled to an increase of axial asymmetry, can explain the XANES changes observed above 7130 eV without an appreciable change of the Fe-N_p distance. Received: 8 October 1997 / Revised version: 10 March 1998 / Accepted: 23 March 1998     

High-resolution A-DNA crystal structures of d(AGGGGCCCCT). An A-DNA model of poly(dG) x poly(dC).

A-DNA conformation is favored by guanine-rich sequences, such as (dG)n x (dC)n, or under low-humidity conditions. Earlier A-DNA crystal structures revealed some conformational variations which may be the result of sequence-dependent effects and/or crystal packing forces. Here we report the high-resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P212121 or the P6122 space group) to gain insights into the conformation and dynamics of the (dG)n x (dC)n sequence. The P212121 form has been analyzed using data to 1.1 A resolution by the anisotropic temperature factor refinement procedure of the SHELX97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A-DNA structure. The backbone torsional angles fall in a narrow range, except for the alpha/gamma angles which have two distinct combinations (gauche-/gauche+ or trans/trans). An A-DNA model of poly(dG) x poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P212121 form. In the crystal structure of the P6122 space group, the central eight base pairs of the decamer adopt A-DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A-DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.     

Three genetic variants of human plasma apolipoprotein A-IV. apoA-IV-1(Thr347----Ser), apoA-IV-0(Lys167----Glu,Gln360----His), and apoA-IV-3(Glu165----Lys).

Human apolipoprotein (apo) A-IV is a genetically polymorphic glyco-protein of 376 residues. We have recently reported the molecular basis for the two most common isoproteins, apoA-IV-1 and apoA-IV-2(Gln360----His), and for two rare variants, apoA-IV-0 (4-amino acid insertion) and apoA-IV-3(Glu230----Lys). In this report, we present the genetic basis for three additional isoproteins of apoA-IV. Sequence analysis of the apoA-IV-1 allele revealed a common nucleotide substitution which converts threonine (ACT), residue 347 of the mature protein, into serine (TCT). In one out of the five subjects with the apoA-IV-1/0 phenotype we identified two point mutations: 1) replacing the positively charged lysine (AAG), amino acid 167, with a negatively charged glutamic acid (GAG), and 2) converting the neutral residue 360, glutamine (CAG), to a positively charged histidine (CAT). We have also characterized four additional heterozygotes for the apoA-IV-3 allele. One individual was found to have the previously described substitution of lysine for glutamic acid at amino acid 230. The three other subjects had the identical mutation but at a different position in the polypeptide chain, residue 165. These results indicate that one predominant allele codes for the isoproteins apoA-IV-2 and apoA-IV-0 and that at least two major allelic variants for the isoproteins apoA-IV-1 and apoA-IV-3 are present in the population analyzed.     

Flow analysis and sorting of microchromosomes (<3 Mb)

BACKGROUND: The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb. METHODS: Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting. RESULTS: Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot. CONCLUSIONS: Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.     

High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide

Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that yield the bioactive ET-1 hormone.     

Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A(2): quaternary structure and inhibition mechanism insights

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. A class of PLA(2)-like proteins has been described which despite PLA(2) activity on artificial substrate, due to a D49K mutation, is still highly myonecrotic. This work reports the X-ray structure determination of two Lys49-PLA(2)s from Bothrops neuwiedi pauloensis (BnSP-7 and BnSP-6) and, for the first time, the comparison of eight dimeric Lys49-PLA(2)s. This comparison reveals that there are not just two ("open" and "closed") but at least six different conformations. The binding of fatty acid observed in three recent Lys49-PLA(2) structures seems to be independent of their quaternary conformation. Cys29 polarization by Lys122 is not significant for BnSP-7 and BnSP-6 or other structures not bound by fatty acids. These structures may be in an "active" state when nothing is bound to them and the Lys122/Cys29 interactions are weak or absent.     

Immunohistochemical analysis of beta3 integrin (CD61): expression in pig tissues and human tumors

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.     

High-resolution X-ray crystal structures of human gammaD crystallin (1.25 A) and the R58H mutant (1.15 A) associated with aculeiform cataract

Several human cataracts have been linked to mutations in the gamma crystallin gene. One of these is the aculeiform cataract, which is caused by an R58H mutation in gammaD crystallin. We have shown previously that this cataract is caused by crystallization of the mutant protein, which is an order of magnitude less soluble than the wild-type. Here, we report the very high-resolution crystal structures of the mutant and wild-type proteins. Both proteins crystallize in the same space group and lattice. Thus, a strict comparison of the protein-protein and protein-water intermolecular interactions in the two crystal lattices is possible. Overall, the differences between the mutant and wild-type structures are small. At position 58, the mutant protein loses the direct ion-pair intermolecular interaction present in the wild-type, due to the differences between histidine and arginine at the atomic level; the interaction in the mutant is mediated by water molecules. Away from the mutation site, the mutant and wild-type lattice structures differ in the identity of side-chains that occupy alternate conformations. Since the interactions in the crystal phase are very similar for the two proteins, we conclude that the reduction in the solubility of the mutant is mainly due to the effect of the R58H mutation in the solution phase. The results presented here are also important as they are the first high-resolution X-ray structures of human gamma crystallins.     

Frequency of common colds and serum levels of sICAM-1 (CD54), sLFA-3 (CD58) and sIL-2R (CD25).

Epidemiologic case-control studies have suggested an inverse relationship between past medical history of frequency of common colds and subsequent cancer risk for various sites. One hypothetical explanation for this finding may be that there are unknown differences in immune responsiveness between patients developing cancer and healthy individuals. The present study examines the relationship between the frequency of common colds and a) plasma levels of sICAM-1/CD54, sLFA-3/CD58 and sIL-2R/CD25 which are believed to modulate activation of immune responses, and b) cell-mediated immunity in vivo as determined by Multitest Mérieux. The investigation was conducted as a correlation study amongst a healthy group of individuals from the general population in Germany. We found a statistically significant inverse relationship between frequency of common colds and levels of sCD58 and, partially, sCD54. No association was found between levels of sCD25 and results of Multitest Mérieux.     

The leukocyte common antigen (CD45) of the Pacific hagfish,Eptatretus stoutii: implications for the primordial function of CD45

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.     

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

X-ray crystal structures of half the human papilloma virus E2 binding site: d(GACCGCGGTC).

The X-ray crystal structure of the DNA decamer d(GACCGCGGTC), containing half the human papilloma virus E2 binding site, has been solved from two crystals grown at different ionic conditions (50 mM MgCl2and 50 mM spermine or 1.56 mM MgCl2and 1.56 mM spermine). Despite the variation in salt concentration, the two DNA structures are in a very similar, A-type DNA conformation, with helical axes curving towards the major groove. Although the salt concentrations do not effect the helical parameters or hydration to a large degree, there is a change in the overall helical curvature; 18 degrees and 31 degrees for the low and high salt structures, respectively. This curvature appears to be sequence specific and biologically relevant when compared with similar DNA structures, including the E2 binding site of a protein-DNA complex.     

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.     

Protonated polynucleotides structures - 22.CD study of the acid-base titration of poly(dG).poly(dC)

The acid-base titration (pH 8 --> pH 2.5 --> pH 8) of eleven mixing curve samples of the poly(dG) plus poly(dC) system has been performed in 0.15 M NaCl. Upon protonation, poly(dG).poly(dC) gives rise to an acid complex, in various amounts according to the origin of the sample. We have established that the hysteresis of the acid-base titration is due to the non-reversible formation of an acid complex, and the liberation of the homopolymers at the end of the acid titration and during the base titration: the homopolymer mixtures remain stable up to pH 7. A 1G:1C stoichiometry appears to be the most probable for the acid complex, a 1G:2C stoichiometry, as found in poly(C(+)).poly(I).poly(C) or poly(C(+)).poly(G).poly(C), cannot be rejected. In the course of this study, evidence has been found that the structural consequences of protonation could be similar for both double stranded poly(dG).poly(dC) and G-C rich DNA's: 1) protonation starts near pH 6, dissociation of the acid complex of poly(dG).poly(dC) and of protonated DNA take place at pH 3; 2) the CD spectrum computed for the acid polymer complex displays a positive peak at 255 nm as found in the acid spectra of DNA's; 3) double stranded poly(dG).poly(dC) embedded in triple-stranded poly(dG).poly(dG).poly(dC) should be in the A-form and appears to be prevented from the proton induced conformational change. The neutral triple stranded poly(dG).poly(dG).poly(dC) appears therefore responsible, although indirectly, for the complexity and variability of the acid titration of poly(dG).poly(dC) samples.     

X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.     

Synthesis and X-ray structures of triphenylphosphine-mercury(II) thiosalicylate complexes: novel aggregation processes

Reaction of [HgCl₂(PPh₃)₂] with one equivalent of thiosalicylic acid (tsalH₂, HSC₆H₄CO₂H) and excess triethylamine, followed by recrystallisation from dichloromethane-diethyl ether gives the compound [Hg₂(tsal)₂(PPh₃)₂] (2). This has a bis(S,O)-chelated mercury centre with a nido-trigonal bipyramidal coodination, with the four oxygens of the two carboxylates also coordinated to a Hg(PPh₃)₂ moiety. When a reduced quantity of pyridine was used as the base a different crystalline product was isolated. This was characterised as [Hg₂(tsal)₂(PPh₃)₂][Hg(tsalH)₂] (3), which contains the same [Hg₂(tsal)₂(PPh₃)₂] moiety found for 2, co-crystallised with a [Hg(tsalH)₂]. The two mercury-thiosalicylate species are connected by means of O-H?O hydrogen bonding.