A major problem in the development of antiviral therapies for AIDS has been the emergence of drug resistance. We report an analysis of the structure of a Val 82 to Ala mutant of HIV-1 proteinase complexed to A-77003, a C2 symmetry-based inhibitor. Modelling studies predicted that the V82A mutation would result in decreased van der Waals' interactions with the phenyl rings of A-77003 in both S1 and S1' subsites. Unexpected rearrangements of the protein backbone, however, resulted in favourable re-packing of inhibitor and enzyme atoms in the S1 but not the S1' subsite. This analysis reveals the importance of enzyme flexibility in accommodating alternate packing arrangements, and can be applied to the re-design of inhibitors targeted to drug resistant variants which emerge in the clinic.

Antiviral Agents↗

PubMed1995

Flap opening in HIV-1 protease simulated by 'activated' molecular dynamics.

We have used an 'activated' molecular dynamics approach to simulate flap opening in HIV-1 protease. An initial impulse for flap opening was provided by applying harmonic constraints to non-flap residues. After an initial 'melting' phase, the two beta-hairpin structures that constitute the flaps opened to a 25 A gap within 200 ps of simulation. Analysis of backbone torsion angles suggests that flap opening is related to conformational changes at Lys 45, Met 46, Gly 52 and Phe 53. In contrast, similar molecular dynamics simulations on the M46I mutant, which is associated with drug resistance, indicates that this mutation stabilizes the flaps in a closed conformation.

Amino Acid Sequence↗

PubMed1995

The not-so-great escape.

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Amino Acid Sequence↗

PubMed1995

Identification and characterization of an outer membrane protein, OmpX, in Escherichia coli that is homologous to a family of outer membrane proteins including Ail of Yersinia enterocolitica.

We previously reported that a region of the Escherichia coli chromosome at 18 min increased E sigma E activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross, Genes Dev. 7:2618-2628, 1993). In the present report, we identify and characterize the gene responsible for the increase in E sigma E activity. This gene is in a monocistronic operon with two promoters and a rho-independent terminator. Sequence analysis of this gene indicated that it encodes an outer membrane protein which is 83% identical to OmpX in Enterobacter cloacae, leading us to name this gene ompX. There are four other proteins that are homologous to OmpX. Several of these proteins, Ail of Yersinia enterocolitica and Rck and PagC of Salmonella typhimurium, have properties that allow bacteria to adhere to mammalian cells, survive exposure to human serum, and/or survive within macrophages. We therefore characterized strains deleted for ompX for their growth phenotypes, E sigma E activity, serum resistance, and adherence to mammalian cells. No differences in growth rates, serum resistance, or adherence to mammalian cells were observed; however, E sigma E activity was dependent on expression of OmpX in certain strain backgrounds.

Amino Acid Sequence↗

PubMed1995

Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor.

Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors.

Amino Acid Sequence↗

PubMed1994

Phosphorescence and optically detected magnetic resonance investigation of the binding of the nucleocapsid protein of the human immunodeficiency virus type 1 and related peptides to RNA.

The RNA and DNA complexes of nucleocapsid protein p7.Zn (NCp7.Zn) of the human immunodeficiency virus type 1 (HIV-1) are studied by phosphorescence and optically detected magnetic resonance (ODMR). The single tryptophan, Trp37, which is located on the C-terminal zinc finger domain is used as an intrinsic probe. Reductions in the triplet state zero-field splitting (zfs) D parameter of Trp37 upon complex formation with poly(I) and poly(U) are observed. These results, in conjunction with the phosphorescence red-shifts and triplet state lifetime reductions that are observed, suggest the presence of aromatic stacking interactions between NCp7.Zn and the bases of the RNA polymers. An alteration of the intersystem crossing pattern upon complex formation, in addition to the above mentioned spectroscopic shifts, also is consistent with previously observed tryptophans that undergo stacking interactions with DNA bases [Zang, L.-H., Maki, A.H., Murphy, J.B., & Chase, J.W. (1987) Biophys. J. 52, 867-872. Tsao, D.H.H., Casas-Finet, J.R., Maki, A.H., & Chase, J.W. (1989) Biophys. J. 55, 927-936]. These conclusions support those from a recent ODMR study [Lam, W.-C., Maki, A.H., Casas-Finet, J.R., Erickson, J.W., Sowder, R.C., II, & Henderson, L.E. (1993) FEBS Lett. 328, 45-48] of NCp7.Zn binding to 5-mercurated polyuridylic acid [poly(5-HgU)] in which stacking interactions between the RNA and NCp7.Zn are inferred from the observation of an external heavy atom effect induced on Trp37.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Aluminum fluoride activation of bovine transducin induces two distinct conformational changes in the alpha subunit.

We have used resonance energy transfer to read out the interactions of the alpha subunit of transducin (alpha T) with the transducin beta gamma subunit complex (beta gamma T) and to compare the rate of aluminum fluoride-induced alpha T activation, as reflected by the enhancement of the alpha T tryptophan fluorescence, with the rate for the dissociation of holotransducin into its component subunits. Specifically, a beta gamma T complex that was labeled with 5-(iodoacetamido)fluorescein (IAF-beta gamma T) served as a donor for resonance energy transfer and an alpha T-GDP species labeled with eosin 5-isothiocyanate (EITC-alpha TGDP) served as the acceptor. The quenching of IAF-beta gamma T fluorescence emission by the addition of the EITC-alpha TGDP species, due to resonance energy transfer between the IAF and EITC moieities, ranged from 10% to 15%. The association of the transducin subunits was rapid (i.e., within the time period of mixing) and dose-dependent, yielding an apparent Kd of approximately 150 nM for the alpha TGDP/beta gamma T interaction. Unexpectedly, we find that the dissociation of IAF-beta gamma T from an aluminum fluoride-activated alpha TGDP/IAF-beta gamma T complex occurs prior to the onset of the intrinsic fluorescence changes in alpha T that accompany activation of this subunit. Thus, there are at least two structural changes in alpha T that result from the occupation of the gamma-phosphate position in the nucleotide binding cleft of alpha T by aluminum fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum Compounds↗

PubMed1994

Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.

Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities.

Amino Acid Sequence↗

PubMed1994

Application of the three-dimensional structures of protein target molecules in structure-based drug design.

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Crystallization↗

PubMed1994

Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms.

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.

Base Sequence↗

PubMed1994

Crystal structure of a tethered dimer of HIV-1 proteinase complexed with an inhibitor.

HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that associate with twofold symmetry. We have determined to 1.8 A the crystal structure of a covalently tethered dimer of HIV PR. The tethered dimer:inhibitor complex is identical in nearly every respect to the complex of the same inhibitor with the wild type dimeric molecule, except for the linker region. Our results suggest that the tethered dimer may be a useful surrogate enzyme for studying the effects of single site mutations on substrate and inhibitor binding as well as on enzyme asymmetry, and for simulating independent mutational drift of the two domains which has been proposed to have led to the evolution of modern day, single-chain aspartic proteinases.

Alcohols↗

PubMed1994

Novel procedure for structure refinement in homology modeling and its application to the human class Mu glutathione S-transferases.

Glutathione S-transferases (GST) are a major class of phase II detoxifying enzymes that conjugate glutathione to electrophiles. Their involvement in the degradation of chemotherapeutic agents, which contributes to drug resistance, makes this family of enzymes potential targets for therapeutic agents. This study generates, by homology modeling, a 3-D structure of three GST human isozymes of the Mu class, M1b-1b, M2-2 and M3-3, using the Rat3-3 GST structure as a template. The high percentage of identity among these enzymes and the lack of insertions and deletions make the system ideally suited to the technique of homology modeling. A novel technique for the modeling of protein structures was applied. The structure of the template was used to generate a low-resolution crystallographic map in which the initial coordinates of the structure to be modeled were placed. The structure was then annealed within this envelope. In addition, a feedback-restrained molecular dynamics procedure was adopted to scale the template restraints during the simulations. Three independent validation procedures were applied. To assess the reliability of the methods, an identical series of simulation steps to those used in the refinement were applied to the template structure (self modeling). Further, a homology structure for the Rat3-3 template was generated, starting from the modeled M1b-1b structure (reverse modeling). To assess the reasonableness of the modeled structures, two recently developed methodologies to verify protein structures based on statistics of the nonbonded interactions were applied. Overall, the structures appear to be consistent.

Amino Acid Sequence↗

PubMed1994

Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor.

Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease.

Amino Acid Sequence↗

PubMed1993

Evidence for stacking interactions between 5-mercurated polyuridylic acid and HIV-1 p7 nucleocapsid protein obtained by phosphorescence and optically detected magnetic resonance (ODMR).

The photoexcited triplet state of Trp-37 in the C-terminal zinc finger of the HIV-1 p7 nucleocapsid protein was used as a probe of p7 interactions with the heavy atom-derivatized RNA homopolymer, poly-5-mercuriuridylic acid (5-HgU). Binding of p7 to 5-HgU (Hg blocked with 2-mercaptoethanol) produces an external heavy atom effect (HAE) on Trp-37 characterized by fluorescence quenching, reduction of the phosphorescence lifetime by three orders of magnitude, and the appearance of the D+E phosphorescence-detected ODMR signal, absent in unperturbed Trp, but induced by a HAE. The details of the HAE are consistent with out-of-plane van der Waals contact of Hg with the indole chromophore of Trp-37. Steric requirements suggest further that the Trp-RNA contact occurs via an aromatic stacking interaction.

Amino Acid Sequence↗