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Biomedical subjects

Jan Pieter Abrahams

Publications and source records attributed to Jan Pieter Abrahams.

At least 19 recordsLinked to original sources
PubMed2006

Structure of the E. coli signal recognition particle bound to a translating ribosome.

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 A structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.

Base Sequence↗
PubMed2006

Nano-dispensing by electrospray for biotechnology.

Liquid transport of minute amounts of biomaterials is of paramount importance in many biotechnological applications. One of the challenges is the transport of viscous liquids without heating. Electro hydro dynamic atomization or electrospray is a viable method for the controlled transport of nanoliter volume of viscous liquids as shown for PEG400. Experimental results and the design of a novel spraying configuration, which can be incorporated in an optical microscope, are reported.

Biotechnology↗
PubMed2005

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 A. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 A for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.

Animals↗
PubMed2004

Crystal structure of an empty capsid of turnip yellow mosaic virus.

Empty capsids (artificial top component) of turnip yellow mosaic virus were co-crystallized with an encapsidation initiator RNA hairpin. No clear density was observed for the RNA, but there were clear differences in the conformation of a loop of the coat protein at the opening of the pentameric capsomer (formed by five A-subunits) protruding from the capsid, compared to the corresponding loop in the intact virus. Further differences were found at the N terminus of the A-subunit. These differences have implications for the mechanism of decapsidation of the virus, required for infection.

Capsid↗
PubMed2004

Crystallization and preliminary X-ray crystallographic studies on a Kunitz-type potato serine protease inhibitor.

Interest in protease inhibitors has been renewed because of their potent activity in preventing carcinogenesis in a wide variety of in vivo and in vitro model systems. Potato tubers contain a wide range of such protease inhibitors. In cv. Elkana potato tubers, protease inhibitors represent about 50% of the total amount of soluble protein. Potato serine protease inhibitor (PSPI), one of the isoforms of the most abundant group of protease inhibitors, is a dimeric double-headed Kunitz-type inhibitor. No high-resolution structural information on this type of inhibitor has so far been obtained, as all currently known structures are of the monomeric single-headed or monomeric double-headed types. Crystals were grown in 0.1 M HEPES pH 7.5, 10% PEG 8000 and 8% ethylene glycol complemented with 9 mM 1-s-octyl-beta-D-thioglucoside or 0.1 M glycine. Data were collected from a single crystal under cryoconditions to 1.8 A resolution. The protein crystallized in space group P2(1), with unit-cell parameters a = 54.82, b = 93.92, c = 55.44 A, beta = 100.7 degrees; the scaling Rsym is 0.044 for 45,456 unique reflections.

Crystallization↗
PubMed2004

Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor.

The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.

Alanine↗
PubMed2004

Structure of an anti-Lewis X Fab fragment in complex with its Lewis X antigen.

The Lewis X trisaccharide is pivotal in mediating specific cell-cell interactions. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, has been shown to recognize the Lewis X trisaccharide. Here we describe the structure of the Fab fragment of 291-2G3-A, with Lewis X, to 1.8 A resolution. The crystallographic analysis revealed that the antigen binding site is a rather shallow binding pocket, and residues from all six complementary determining regions of the antibody contact all sugar residues. The high specificity of the binding pocket does not result in high affinity; the K(D) determined by isothermal calorimetry is 11 microM. However, this affinity is in the same range as for other sugar-antibody complexes. The detailed understanding of the antibody-Lewis X interaction revealed by the crystal structure may be helpful in the design of better diagnostic tools for schistosomiasis and for studying Lewis X-mediated cell-cell interactions by antibody interference.

Base Sequence↗
PubMed2004

CRANK: new methods for automated macromolecular crystal structure solution.

CRANK is a novel suite for automated macromolecular structure solution and uses recently developed programs for substructure detection, refinement, and phasing. CRANK utilizes methods for substructure detection and phasing and combines them with existing crystallographic programs for density modification and automated model building in a convenient and easy-to-use CCP4i graphical interface. The data model used conforms to the XML eXtensible Markup Language specification and works as a common language to communicate data between many different applications inside and outside of the suite. The application of CRANK on various test cases has yielded promising results: with minimal user input, CRANK can produce better quality solutions over currently available programs.

Crystallography↗
PubMed2003

Visualization by cryo-electron microscopy of genomic RNA that binds to the protein capsid inside bacteriophage MS2.

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.

Capsid Proteins↗
PubMed2003

Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion.

Apoptin, a protein derived from chicken anemia virus, induces apoptosis in human transformed or tumor cells but not in normal cells. When produced in bacteria as a recombinant fusion with maltose-binding protein (MBP-Apoptin), Apoptin forms a distinct, stable multimeric complex that is remarkably homogeneous and uniform. Here, using cytoplasmic microinjection, we showed that recombinant MBP-Apoptin multimers retained the characteristics of the ectopically expressed wild-type Apoptin; namely, the complexes translocated to the nucleus of tumor cells and induced apoptosis, whereas they remained in the cytoplasm of normal, primary cells and exerted no apparent toxic effect. In normal cells, MBP-Apoptin formed increasingly large, organelle-sized globular bodies with time postinjection and eventually lost the ability to be detected by immunofluorescence analysis. Costaining with an acidotrophic marker indicated that these globular structures did not correspond to lysosomes. Immunoprecipitation studies showed that MBP-Apoptin remained fully antibody-accessible regardless of buffer stringency when microinjected into tumor cells. In contrast, MBP-Apoptin in normal cells was only recoverable under stringent lysis conditions, whereas under milder conditions they became fully shielded with time on two epitopes spanning the entire protein. Further biochemical analysis showed that the long-term fate of Apoptin protein aggregates in normal cells was their eventual elimination. Our results provide the first example of a tumor-specific apoptosis-inducing aggregate that is essentially sequestered by factors or conditions present in the cytoplasm of healthy, nontransformed cells. This characteristic should reveal more about the cellular interactions of this viral protein as well as further enhance its safety as a potential tumor-specific therapeutic agent.

Antineoplastic Agents↗
PubMed2003

Apoptin protein multimers form distinct higher-order nucleoprotein complexes with DNA.

The chicken anaemia virus-derived protein apoptin is a tumour-specific cell-killing agent. It is biologically active as a highly stable, multimeric complex, consisting of 30-40 monomers. In tumour cells, but negligibly in normal cells, apoptin is imported into the nucleus prior to the induction of apoptosis. Immunoelectron microscopic data we report here indicate that apoptin predominantly co-localises with heterochromatin and nucleoli within tumour cells. Apoptin's preference for these DNA-dense nuclear bodies may be explained by our finding that apoptin cooperatively forms distinct superstructures with DNA in vitro. These superstructures do not grow beyond a diameter of approximately 200 nm, containing up to 20 multimeric apoptin complexes and approximately 3 kb of DNA. Furthermore, we show a single apoptin multimer to have eight independent, non-specific DNA-binding sites which preferentially bind strand ends, but which can also collaborate to bind longer stretches of DNA. Apoptin's high affinity for naked, undecorated double- and single-stranded DNA and for DNA fibre ends suggests that it may also capture such DNA in superstructures in vivo. Since these forms of DNA are predominantly found in transcriptionally active, replicating and damaged DNA, apoptin could be triggering apoptosis by interfering with DNA transcription and synthesis.

Binding Sites↗
PubMed2003

The structure of the receptor-binding domain of the bacteriophage T4 short tail fibre reveals a knitted trimeric metal-binding fold.

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipo-polysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube. The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12).X-ray crystallography at 1.5A resolution of a protease-stable fragment of gp12 generated in the presence of zinc chloride reveals the structure of the C-terminal receptor-binding domain. It has a novel "knitted" fold, consisting of three extensively intertwined monomers. It reveals a metal-binding site, containing a zinc ion coordinated by six histidine residues in an octahedral conformation. We also suggest an LPS-binding region.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary X-ray analysis of an anti-LewisX Fab fragment with and without its LewisX antigen.

LewisX-containing glycoconjugates are abundantly expressed by schistosomes and are assumed to be of prime importance for the survival of the parasite within the human host. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, was found to interact with monomers, dimers and trimers of the LewisX trisaccharide. The Fab fragment of monoclonal antibody 291-2G3-A has been crystallized and soaked with its LewisX antigen. X-ray data sets were recorded for the different Fab crystals with and without LewisX. Crystals grown from 25% polyethylene glycol 3350, 0.17 M ammonium sulfate and 15% glycerol belong to the triclinic space group P1, with unit-cell parameters a = 67.4, b = 71.6, c = 104.8 A, alpha = 86.5, beta = 71.3, gamma = 83.3 degrees for the native crystals and with slightly different unit-cell parameters a = 67.3, b = 72.4, c = 104.8 A, alpha = 85.8, beta = 71.3, gamma = 83.3 degrees for the crystals containing bound LewisX. Crystals grown from 14% PEG 3350, 50 mM Tris pH 8 and soaked with LewisX also belong to the triclinic space group P1, but with different unit-cell parameters a = 45.1, b = 60.8, c = 91.6 A, alpha = 96.0, beta = 95.4, gamma = 101.8 degrees.

Animals↗
PubMed2003

The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain, as revealed by a pathological mutant.

C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.

Amino Acid Sequence↗
PubMed2003

Structure of beta-antithrombin and the effect of glycosylation on antithrombin's heparin affinity and activity.

Antithrombin is a member of the serpin family of protease inhibitors and the major inhibitor of the blood coagulation cascade. It is unique amongst the serpins in that it circulates in a conformation that is inactive against its target proteases. Activation of antithrombin is brought about by a conformational change initiated upon binding heparin or heparan sulphate. Two isoforms exist in the circulation, alpha-antithrombin and beta-antithrombin, which differ in the amount of glycosylation present on the polypeptide chain; beta-antithrombin lacks the carbohydrate present at Asn135 in alpha-antithrombin. Of the two forms, beta-antithrombin has the higher affinity for heparin and thus functions as the major inhibitor in vivo even though it is the less abundant form. The reason for the differences in heparin affinity between the alpha and beta-forms have been shown to be due to the additional carbohydrate changing the rate of the conformational change. Here, we describe the most accurate structures of alpha-antithrombin and alpha-antithrombin+heparin pentasaccharide reported to date (2.6A and 2.9A resolution, respectively, both re-refinements using old data), and the structure of beta-antithrombin (2.6A resolution). The new structures have a remarkable degree of ordered carbohydrate and include parts of the antithrombin chain not modeled before. The structures have allowed a detailed comparison of the conformational differences between the three. They show that the structural basis of the lower affinity for heparin of alpha-antithrombin over beta-antithrombin is due to the conformational change that occurs upon heparin binding being sterically hindered by the presence of the additional bulky carbohydrate at Asn135.

Antithrombins↗
PubMed2003

ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase.

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat alpha1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the gamma-phosphate group to the invariant Asp369.

Adaptor Protein Complex 2↗