Cation binding to the integrin CD11b I domain and activation model assessment.

BACKGROUND: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11 b I domain and to evaluate the structural effects of divalent ion binding to this protein. RESULTS: We have determined the X-ray structure of a new crystal form of the CD11 b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straight-forward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11 a I-domain structures and a CD11 b I-domain complex with Mn2+. These structures define a majority conformation. CONCLUSIONS: Addition of the cations Mg2+, Mn2+ and Cd2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al, is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.

Recombinant human cytomegalovirus protease with a C-terminal (His)6 extension: purification, autocatalytic release of the mature enzyme, and biochemical characterization.

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli. The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E. coli as inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E. coli. In addition, the refolded CMV PR could be crystallized for X-ray diffraction.

Fluorescence and site-directed mutagenesis studies of interleukin 1 beta.

The authors mutated two key residues in the sequence of the cytokine interleukin 1 beta, namely the double mutant Phe46 to Trp46 and Trp120 to Phe120 and the single point mutation Lys103 to Leu103 and measured the resulting receptor binding and biological activities. The biological and receptor binding activities of the Trp46 mutein was reduced by a factor of 12 and 25, respectively, and surprisingly, those of the Leu103 mutein, 2600 and 600-fold relative to the wild-type protein. The authors had previously showed that Lys103 was unusually reactive to a variety of derivatizing agents. Furthermore, the Trp to Phe mutation allowed us to monitor the local environment of that residue by studying its intrinsic fluorescence properties, as well as any change in the fluorescence properties of Trp120 of the Leu103 mutein. The results of these studies show that mutation of Lys103 to Leu103 produces subtle long-range changes in the micro-environment of Trp120, indicative of a key role for this residue in the folding of the entire protein.

The crystallographic structure of the protease from human immunodeficiency virus type 2 with two synthetic peptidic transition state analog inhibitors.

The crystal structure of human immunodeficiency virus (HIV) type 2 protease has been determined in complexes with peptidic inhibitors Noa-His-Cha psi [CH(OH)CH(OH)]Val-Ile-Amp (U75875) and Qnc-Asn-Cha psi [CH(OH)CH2]Val-Npt(U92163) (where Noa is naphthyloxyacetyl, Cha is cyclohexylalanine, Amp is 2-aminomethylpyridine, Qnc is quinoline-2-carbonyl, and Npt is neopentylamine), which have dihydroxyethylene and hydroxyethylene moieties, respectively, in place of the normal scissile bond of the natural ligand. The complexes crystallize in space group P2(1)2(1)2(1), with one dimer-inhibitor complex per asymmetric unit and average cell dimensions of a = 33.28 A, b = 45.35 A, c = 135.84 A. Data were collected to approximately 2.5-A resolution. The model structures were refined with resulting R-factors of around 0.19. As expected, the HIV-2 protease structure is approximately C2-symmetric with a gross structure very similar to that of the HIV-1 enzyme. The inhibitors bind in an extended conformation positioned lengthwise in the binding cleft in a manner similar to that found in the HIV-1 protease-inhibitor complexes previously reported. The substitution of the bulkier Ile82 side chain in the HIV-2 protease may help explain the better ability of HIV-2 protease to bind and hydrolyze ligands with small P1 and P1' side groups. It appears that differences in specificity between the proteases of HIV-1 and HIV-2 are not merely a result of simple side chain substitutions, but may be complicated by differences in main chain flexibility as well.

Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides.

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.

A two step purification of recombinant human interleukin-1 beta expressed in E. coli.

Recombinant human interleukin-1 beta has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1 beta is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1 beta (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 x 10(7) units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1 beta sera.

Effect of ribosome binding site on gene expression in Escherichia coli.

Using the expression of human renin gene in Escherichia coli as a model, we have observed that a distance of 7-10 nucleotides between the Shine-Dalgarno site to the initiation codon ATG is optimal for translation initiation. The sequence ATA as the triplet preceding the ATG gives the best expression among those tested. These observations agree with the statistical bias observed for the genes of E. coli and its phages. We have also compared gene expression with three different Shine-Dalgarno sites. Expression of a given gene can be increased by as much as 1000-fold with slight modifications in the Shine-Dalgarno sequence.

Crystallization of purified recombinant human interleukin-1 beta.

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.

Re-evaluation of selectivity of malachite green test for presence of sulfite in mechanically deboned poultry.

A study was undertaken to determine whether free chlorine or sulfide anions could yield positive results for the malachite green test in meat samples when sulfite was not present. Malachite green solution was added to a mechanically deboned poultry (MDP) sample spiked with either chlorine or sulfide ions. The presence of chlorine in MDP samples at any concentration tested did not yield a positive reaction for the malachite green test. When sulfide ions were introduced into MDP samples, positive results were observed at 40 micrograms sulfide ion/g MDP and above. Because aqueous solutions of sulfide at the levels mentioned have a relatively high pH, it was necessary to determine whether the pH or the presence of the sulfide ions had caused this positive reaction to the malachite green test. After a comparison of a solution of sulfide, and a solution of a matching pH, it was observed that the sulfide present caused the positive reaction to the malachite green test.

Titrimetric determination of calcium in mechanically separated poultry and beef: collaborative study.

A collaborative study was conducted on a titrimetric EDTA method for the determination of calcium in mechanically separated poultry and beef tissue. Six laboratories analyzed, as blind duplicates, 5 poultry samples and 5 beef samples. Calcium concentrations covered the levels from 0.04 to 0.82%. Pooled standard deviations were 0.0165 for repeatability, 0.0072 for bias, and 0.0225 for reproducibility. The method has been adopted official first action.