Structure of an EF-Tu complex with a thiazolyl peptide antibiotic determined at 2.35 A resolution: atomic basis for GE2270A inhibition of EF-Tu.

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.

Effect of protease inhibitors on the sense of taste.

The purpose of this study was to investigate the taste properties of protease inhibitors which are essential components of drug regimes used to treat human immunodeficiency virus (HIV) infection. In this study, the taste properties of four protease inhibitors (indinavir, ritonavir, saquinavir, and nelfinavir) were investigated in unmedicated HIV-infected patients and healthy controls. Three of the four protease inhibitors (indinavir, ritonavir, and saquinavir) were found to be predominantly bitter (with additional qualities of medicinal, metallic, astringent, sour, and burning). Nelfinavir was found to be relatively tasteless. HIV-infected and uninfected control subjects detected protease inhibitors at similar concentrations, but HIV-infected subjects perceived suprathreshold concentrations as more bitter than controls. Detection thresholds ranged from 0.0061 mM for saquinavir in HIV-infected patients to 0.0702 mM for ritonavir in uninfected control subjects. Suprathreshold studies indicated that protease inhibitors modified the taste perception of a variety of other taste compounds. These results are consistent with clinical findings that protease inhibitors produce taste complaints that can impact patient compliance.

Sequence profile of the parallel beta helix in the pectate lyase superfamily.

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.

Structure-based multiple alignment of extracellular pectate lyase sequences.

Pectate lyases are secreted virulence factors which degrade the pectate component of plant cell walls. The evolutionary-based multiple alignment of extracellular pectate lyases has been corrected using three-dimensional structural information derived from Erwinia chyrsanthemi pectate lyases C and E. The new multiple alignment reveals invariant amino acids likely to be involved in two different enzymatic functions.

Functional implications of structure-based sequence alignment of proteins in the extracellular pectate lyase superfamily.

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu.

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.