A multi-drug resistant HIV-1 protease is resistant to the dimerization inhibitory activity of TLF-PafF.

Human immunodeficiency virus type-1 (HIV-1) protease, a homodimeric aspartyl protease, is a critical drug target in designing anti-retroviral drugs to treat HIV/AIDS. Multidrug-resistant (MDR) clinical isolate-769 HIV-1 protease (PDB ID: 3PJ6) has been shown to exhibit expanded active site cavity with wide-open conformation of flaps (Gly48-Gly52) due to the accumulation of multiple mutations. In this study, an HIV-1 protease dimerization inhibitor (PDI)-TLF-PafF, was evaluated against MDR769 HIV-1 protease using X-ray crystallography. It was hypothesized that co-crystallization of MDR769 HIV-1 protease in complex with TLF-PafF would yield either a monomeric or a disrupted dimeric structure. However, crystal structure of MDR769 I10V HIV-1 protease co-crystallized with TLF-PafF revealed an undisrupted dimeric protease structure (PDB ID: 4NKK) that is comparable to the crystal structure of its corresponding apo-protease (PDB ID: 3PJ6). In order to understand the binding profile of TLF-PafF as a PDI, docking analysis was performed using monomeric protease (prepared from the dimeric crystal structure, PDB ID: 4NKK) as docking receptor. Docking analysis revealed that TLF-PafF binds at the N and C termini (dimerization domain) in a clamp shape for the monomeric wild type receptor but not the MDR769 monomeric receptor. TLF-PafF preferentially showed higher binding affinity to the expanded active site cavity of MDR769 HIV-1 protease than to the termini. Irrespective of binding location, the binding affinity of TLF-PafF against wild type receptor (-6.7kcal/mol) was found to be higher compared to its corresponding binding affinity against MDR receptor (-4.6kcal/mol) suggesting that the MDR769 HIV-1 protease could be resistant to the PDI-activity of TLF-PafF, thus supporting the dimeric crystal structure (PDB ID: 4NKK).

Contribution of the 80s loop of HIV-1 protease to the multidrug-resistance mechanism: crystallographic study of MDR769 HIV-1 protease variants.

The flexible flaps and the 80s loops (Pro79-Ile84) of HIV-1 protease are crucial in inhibitor binding. Previously, it was reported that the crystal structure of multidrug-resistant 769 (MDR769) HIV-1 protease shows a wide-open conformation of the flaps owing to conformational rigidity acquired by the accumulation of mutations. In the current study, the effect of mutations on the conformation of the 80s loop of MDR769 HIV-1 protease variants is reported. Alternate conformations of Pro81 (proline switch) with a root-mean-square deviation of 3-4.8 Å in the C(α) atoms of the I10V mutant and a side chain with a `flipped-out' conformation in the A82F mutant cause distortion in the S1/S1' binding pockets that affects inhibitor binding. The A82S and A82T mutants show local changes in the electrostatics of inhibitor binding owing to the mutation from nonpolar to polar residues. In summary, the crystallographic studies of four variants of MDR769 HIV-1 protease presented in this article provide new insights towards understanding the drug-resistance mechanism as well as a basis for design of future protease inhibitors with enhanced potency.

"Wide-open" 1.3 A structure of a multidrug-resistant HIV-1 protease as a drug target.

This report examines structural changes in a highly mutated, clinical multidrug-resistant HIV-1 protease, and the crystal structure has been solved to 1.3 A resolution in the absence of any inhibitor. This protease variant contains codon mutations at positions 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90 that confer resistance to protease inhibitors. Major differences between the wild-type and the variant include a structural change initiated by the M36V mutation and amplified by additional mutations in the flaps of the protease, resulting in a "wide-open" structure that represents an opening that is 8 A wider than the "open" structure of the wild-type protease. A second structural change is triggered by the L90M mutation that results in reshaping the 23-32 segment. A third key structural change of the protease is due to the mutations from longer to shorter amino acid side chains at positions 82 and 84.

Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity.

The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (A) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease "flaps" stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 A. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k(off) rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k(on) and k(off) data (K(d) = k(off)/k(on)) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

HIV-1 protease variants from 100-fold drug resistant clinical isolates: expression, purification, and crystallization.

High-resolution X-ray crystallographic structures of HIV-1 protease clinical variants complexed with licensed inhibitors are essential to understanding the fundamental cause of protease drug resistance. There is a need for structures of naturally evolved HIV-1 proteases from patients failing antiretroviral therapy. Here, we report the expression, purification, and crystallization of clinical isolates of HIV-1 protease that have been characterized to be more than 100 times less susceptible to US FDA approved protease inhibitors.

Pseudomonas aeruginosa aspartate transcarbamoylase. Characterization of its catalytic and regulatory properties.

Aspartate transcarbamoylase from Pseudomonadaceae is a class A enzyme consisting of six copies of a 36-kDa catalytic chain and six copies of a 45-kDa polypeptide of unknown function. The 45-kDa polypeptide is homologous to dihydroorotase but lacks catalytic activity. Pseudomonas aeruginosa aspartate transcarbamoylase was overexpressed in Escherichia coli. The homogeneous His-tagged protein isolated in high yield, 30 mg/liter of culture, by affinity chromatography and crystallized. Attempts to dissociate the catalytic and pseudo-dihydroorotase (pDHO) subunits or to express catalytic subunits only were unsuccessful suggesting that the pDHO subunits are required for the proper folding and assembly of the complex. As reported previously, the enzyme was inhibited by micromolar concentrations of all nucleotide triphosphates. In the absence of effectors, the aspartate saturation curves were hyperbolic but became strongly sigmoidal in the presence of low concentrations of nucleotide triphosphates. The inhibition was unusual in that only free ATP, not MgATP, inhibits the enzyme. Moreover, kinetic and binding studies with a fluorescent ATP analog suggested that ATP induces a conformational change that interferes with the binding of carbamoyl phosphate but has little effect once carbamoyl phosphate is bound. The peculiar allosteric properties suggest that the enzyme may be a potential target for novel chemotherapeutic agents designed to combat Pseudomonas infection.

Cloning, expression and preliminary X-ray analysis of the dihydroorotase from the hyperthermophilic eubacterium Aquifex aeolicus.

Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway. The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced. The recombinant protein was expressed at high levels and could be purified readily in a single step by Ni(2+) affinity chromatography. Both native and selenomethionine-labeled proteins were crystallized using the hanging-drop vapor-diffusion technique. Screens of the purified protein identified several conditions that yielded crystals; however, the best crystals were obtained using 1 M Li(2)SO(4), 10 mM NiCl(2), 100 mM Tris acetate pH 8.5 as the precipitant. Well formed diamond-shaped crystals appeared within 1 d and continued to grow over several weeks to about 0.5 mm in the largest dimension. The crystals diffract to 1.7 A and belong to space group C2, with unit-cell parameters a = 119.8, b = 88.0, c = 55.2 A, beta = 99.0 degrees and a mosaic spread of 0.6 degrees. There is one DHOase monomer in the asymmetric unit.