Inhibiting protein-protein interactions: a model for antagonist design.

Protein-protein interactions (PPI) are a ubiquitous mode of transmitting signals in cells and tissues. We are testing a stepwise, generic, structure-driven approach for finding low molecular weight inhibitors of protein-protein interactions. The approach requires development of a high-affinity, single chain antibody directed specifically against the interaction surface of one of the proteins to obtain structural information on the interface. To this end, we developed a single chain antibody (sc1E3) against hIL-1beta that exhibited the equivalent affinity of the soluble IL-1 receptor type I (sIL-1R) for hIL-1beta and competitively blocked the sIL-1R from binding to the cytokine. The antibody proved to be more specific for hIL-1beta than the sIL-1R in that it failed to bind to either murine IL-1beta or human/murine IL-1alpha proteins. Additionally, failure of sc1E3 to bind to several hIL-1beta mutant proteins, altered at receptor site B, indicated that the antibody interacted preferentially with this site. This, coupled with other surface plasmon resonance and isothermal titration calorimetry measurements, shows that sc1E3 can achieve comparable affinity of binding hIL-1beta as the receptor through interactions at a smaller interface. This stable single chain antibody based heterodimer has simplified the complexity of the IL-1/IL-1R PPI system and will facilitate the design of the low molecular weight inhibitors of this interaction.

Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins.

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.

Site-directed double fluorescent tagging of human renin and collagenase (MMP-1) substrate peptides using the periodate oxidation of N-terminal serine. An apparently general strategy for provision of energy-transfer substrates for proteases.

Periodate in neutral aqueous solution rapidly converts N-terminal Ser or Thr to an alpha-N-glyoxylyl moiety that can serve as the locus for incorporation of a modifying group [Geoghegan, K. F., and Stroh, J. G. (1992) Bioconjugate Chem. 3, 138-146. Gaertner, H. F. et al. (1992) Bioconjugate Chem. 3, 262-268]. The usefulness of this procedure has been further illuminated in a route to "energy-transfer" substrates for endoproteases. Each such substrate is an oligopeptide cleavable by a proteinase, but modified (usually at its termini) with two chromophores that form an energy donor-acceptor pair. Production of these substrates is an exercise in double site-directed peptide modification. The new route is composed of three steps, beginning from an unprotected peptide in which a sequence recognized by the pertinent enzyme is placed between N-terminal Ser and C-terminal Lys. Lys may not occur elsewhere in the peptide. Periodate oxidation converts the N-terminal Ser to an alpha-N-glyoxylyl group, which is then allowed to form a hydrazone with the carbohydrazide derivative Lucifer Yellow CH, a hydrophilic fluor with a large Stokes shift (excitation maximum, 425 nm; emission maximum, 525 nm). Finally, the modified peptide is allowed to react with 5-carboxytetramethylrhodamine succinimidyl ester. This reaction selectively modifies the epsilon-amino group of C-terminal Lys, the only amino group remaining in the peptide. 5-Carboxytetramethylrhodamine strongly (> 90%) quenches Lucifer Yellow fluorescence by resonance energy transfer in the intact substrate, but enzyme-catalyzed cleavage eliminates the quenching. The resulting increase in fluorescence may be used to follow the hydrolytic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of biological activity of murine recombinant interleukin-1 beta by selective deamidation at asparagine-149.

A biologically active preparation of murine recombinant interleukin-1 beta (mIL-1 beta) from Escherichia coli cell lysates contained tow forms of mIL-1 beta with pI 8.7 and pI 8.1, respectively. Treatment with 0.1 M Tris, pH 8.5, at 37 degrees C for 35 h converted the pI 8.7 form to the pI 8.1 form by the selective deamidation of an asparagine residue (Asn149) in the mIL-1 beta molecule. Deamidated mIL-1 beta had 3- to 5-fold lower co-mitogenic activity and receptor affinity than the unmodified form.

Isolation and characterization of biologically active murine interleukin-1 alpha derived from expression of a synthetic gene in Escherichia coli.

A murine interleukin-1 alpha (mIL-1 alpha) gene coding for amino acids 115 to 270 of the precursor protein (Lomedico, P.T., Gubler, U., Hellmann, C.P., Dukovich, M., Giri, J.G., Pan, Y.E., Collier, K., Semionow, R., Chua, A.O. and Mizel, S.B. (1984) Nature 312, 458-462) was chemically synthesized and expressed in Escherichia coli. mIL-1 alpha, in the form of insoluble inclusion bodies, accounted for approx. 30% of total cellular protein produced by the recombinant strain. A simple isolation protocol was developed in which inclusion body material was first solubilized in 3 M guanidine hydrochloride, and the mIL-1 alpha was then simultaneously purified and allowed to fold to its active conformation by dialysis against distilled water. This procedure yielded pure, biologically active mIL-1 alpha with 41% recovery of the mIL-1 alpha present in the guanidine hydrochloride extract. The purified preparation had the expected amino acid composition, a molar absorptivity of 28,200 M-1.cm-1 and a pI of 5.2. No methionyl-mIL-1 alpha was detected by N-terminal sequence analysis, and the endotoxin level was less than 10 pg per micrograms of mIL-1 alpha. The specific biological activity was 3.10(7) units/mg in a co-mitogenic thymocyte proliferation assay. In addition to full-length mIL-1 alpha, the preparation contained N-terminally truncated mIL-1 alpha species (mainly des-4 and des-6 amino acid forms). The truncated species were isolated and found to have the same biological activity as the complete polypeptide. Thus, the active fragment of mIL-1 alpha appears to consist of a proteinase-sensitive N-terminal region which is not essential for activity, and a proteinase-resistant core which harbors the essential determinants of its cytokine function.

The use of high field NMR to determine homogeneity in a preparation of human C5a produced by Escherichia coli.

The polypeptide backbone of human C5a was prepared by recombinant DNA techniques. Standard biochemical analysis guided the protein separation to give a sample of C5a which was deemed homogeneous. However, nuclear magnetic resonance (NMR) studies showed the material to be significantly heterogeneous. Reanalysis by high performance liquid chromatography (HPLC) corroborated the NMR results. Further separation by HPLC and analysis by NMR spectroscopy guided the isolation of rC5a to greater than 92% purity. NMR analysis, immunochemical and biological evaluation of the impurities showed them to be C5a structural variants. These results indicate that conventional methods of protein chemistry can fail to reveal heterogeneity in recombinant proteins, and in some circumstances NMR spectroscopy can aid in their purification.

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Experimental evolution of penicillin G acylases from Escherichia coli and Proteus rettgeri.

Proteus rettgeri and Escherichia coli W were shown to express structurally different penicillin G acylases. The enzymes had similar substrate specificity but differed in molecular weight, isoelectric point, and electrophoretic mobility in polyacrylamide gels and did not antigenically cross-react. When the organisms were subjected to environmental conditions which made expression of this enzyme essential for growth, spontaneous mutants were isolated that used different amides as the only source of nitrogen. These mutants acquired the ability to use amides for growth by deregulating the penicillin G acylase and by their evolution to novel substrate specificities. The enzymes expressed by mutants isolated from each genus appeared to have evolved in parallel since each acylase attained similar new substrate specificities when the organisms were subjected to identical selection pressure.

Role of protein subunits in Proteus rettgeri penicillin G acylase.

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.

Repression of penicillin G acylase of Proteus rettgeri by tricarboxylic acid cycle intermediates.

The regulation of the penicillin acylase in proteus rettgeri ATCC 31052 was compared with that of the enzyme in Escherichia coli ATCC 9637. Unlike the E. coli acylase, the P. rettgeri enzyme was not induced by phenylacetic acid, nor was it subject to catabolite repression by glucose. The P. rettgeri acylase appears to be expressed constitutively but is subject to repression by the C4-dicarboxylic acids of the tricarboxylic acid cycle, succinate, fumarate, and malate.

Induction of 3-hydroxybenzoate 2-hydroxylase in a Pseudomonas testosteroni mutant.

Benzoate was established as the inducer of a unique 3-hydroxybenzoate 2-hydroxylase activity found in a Pseudomonas testosteroni mutant which is unable to grow on m-hydroxybenzoate as its sole source of carbon and energy.

Bioconversion of m-hydroxybenzoate to 2,3-dihydroxybenzoate by mutants of Pseudomonas testosteroni.

Mutans of Pseudomonas testosteroni were isolated for their inability to grow on m-hydroxybenzoate as sole carbon source. These mutants hydroxylated m-hydroxybenzoate for form 2,3-dihydroxybenzoate in high yeilds. The bioconversion described in this report represents the first reported example of 3-hydroxybenzoate 2-hydroxylase activity.