Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref -1)

The structure of a 1:1 molar complex between Escherichia coli elongation factor (EF) Tu-GDP and the cyclic thiazolyl peptide antibiotic, GE2270A, has been determined by X-ray diffraction analysis to a resolution of 2.35 A and refined to a crystallographic refinement factor of 20.6%. The antibiotic binds in the second domain of EF-Tu-GDP, making contact with three segments of amino acids (residues 215-230, 256-264, and 273-277). The majority of the protein-antibiotic contacts are van der Waals interactions. A striking feature of the antibiotic binding site is the presence of a salt bridge, not previously observed in other EF-Tu complexes. The ionic interaction between Arg 223 and Glu 259 forms over the antibiotic and probably accounts for the strong affinity observed between EF-Tu and GE2270A. Arg 223 and Glu 259 are highly conserved, but not invariant throughout the prokaryotic EF-Tu family, suggesting that the antibiotic may bind EF-Tu from some organisms better than others may. Superposition of the antibiotic binding site on the EF-Tu-GTP conformation reveals that one region of the antibiotic would form steric clashes with the guanine nucleotide-binding domain in the GTP, but not the GDP, conformation. Another region of the antibiotic binds to the same site as the aminoacyl group of tRNA. Together with prior biochemical studies, the structural findings confirm that GE2270A inhibits protein synthesis by blocking the GDP to GTP conformational change and by directly competing with aminoacyl-tRNA for the same binding site on EF-Tu. In each of the bacterial strains that are resistant to GE2270A, the effect of a site-specific mutation in EF-Tu could explain resistance. Comparison of the GE2270A site in EF-Tu with sequence homologues, EF-G and EF-1alpha, suggests steric clashes that would prevent the antibiotic from binding to translocation factors or to the eukaryotic equivalent of EF-Tu. Although GE2270A is a potent antibiotic, its clinical efficacy is limited by its low aqueous solubility. The results presented here provide the details necessary to enhance the solubility of GE2270A without disrupting its inhibitory properties.

Pels (EC 4.2.2.2) are depolymerizing enzymes that degrade the PGA component of plant cell walls, causing tissue maceration and cell death. The enzymes are normally secreted by phytopathogenic organisms and are the causative agents of virulence in "soft rot" diseases caused by Erwinia sp. (Collmer and Keen, 1986;Kotoujansky, 1987). By sequence analysis, Pels belong to four distinct subfamilies, only two of which appear to be evolutionarily and structurally related. The two related subfamilies, pel-ADE and peZBC, are classified according to various properties, including pI and number of disulfide bonds. Both share three sequence patterns, AxDIKGxxxxVTxS, VxxRxPxxRxGxxHxxxN, and vWiDH (Tamaki et al., 1988;Hinton et al., 1989;Hugouvieux-Cotte-Pattat and Robert-Baudouy, 1992;Barras et al., 1994), which are used to characterize a The authors gratefully acknowledge the support of the U.S. Department of ) and the San Diego Supercomputer Center.

The parallel beta helix structure found in the pectate lyase superfamily has been analyzed in detail. A comparative analysis of known structures has revealed a unique sequence profile, with a strong positional preference for specific amino acids oriented toward the interior of the parallel beta helix. Using the unique sequence profile, search patterns have been constructed and applied to the sequence databases to identify a subset of proteins that are likely to fold into the parallel beta helix. Of the 19 families identified, 39% are known to be carbohydrate-binding proteins, and 50% belong to a broad category of proteins with sequences containing leucine-rich repeats (LRRs). The most striking result is the sequence match between the search pattern and four contiguous segments of internalin A, a surface protein from the bacterial pathogen Listeria monocytogenes. A plausible model of the repetitive LRR sequences of internalin A has been constructed and favorable 3D-1D profile scores have been calculated. Moreover, spectroscopic features characteristic of the parallel beta helix topology in the pectate lyases are present in the circular dichroic spectrum of internalin A. Altogether, the data support the hypothesis that sequence search patterns can be used to identify proteins, including a subset of LRR proteins, that are likely to fold into the parallel beta helix.

Two crystal forms of a complex between trypsin-modified elongation factor Tu-MgGDP from Escherichia coli and the antibiotic tetracycline have been solved by X-ray diffraction analysis to resolutions of 2.8 and 2.1 A, respectively. In the P2(1) form, cocrystals were grown from a solution mixture of the protein and tetracycline. Six copies of the trypsin-modified EF-Tu-MgGDP-tetracycline complex are arranged as three sets of dimers in the asymmetric unit. In the second crystal form, tetracycline was diffused into P4(3)2(1)2 crystals, resulting in a monomeric complex in the asymmetric unit. Atomic coordinates have been refined to crystallographic R factors of 18.0% for the P2(1) form and 20.0% for the P4(3)2(1)2 form. In both complexes, tetracycline makes significant interactions with the GTPase active site of EF-Tu. The phenoldiketone moiety of tetracycline interacts directly with the Mg(2+), the alpha-phosphate group of GDP and two amino acids, Thr25 and Asp80, which are conserved in the GX(4)GKS/T and DX(2)G sequence motifs found in all GTPases and many ATPases. The molecular complementarity, previously unrecognized between invariant groups present in all GTPase/ATPases and the active moiety of tetracycline, may have wide-ranging implications for all drugs containing the phenoldiketone moiety as well as for the design of new compounds targeted against a broad range of GTPases or ATPases.