Kinetic Evidence Supports the Existence of Two Halide Binding Sites that Have a Distinct Impact on the Heme Iron Microenvironment in Myeloperoxidase †

ABSTRACT: Myeloperoxidase (MPO) structural analysis has suggested that halides and pseudohalides bind to the distal binding site and serve as substrates or inhibitors, while others have concluded that there are two separate sites. Here, evidence for two distinct binding sites for halides comes from the bell-shaped effects observed when the second-order rate constant of nitric oxide (NO) binding to MPO was plotted versus Cl -concentration. The chloride level used in the X-ray structure that produced Cl -binding to the amino terminus of the helix halide binding site was insufficient to populate either of the two sites that appear to be responsible for the two phases. Biphasic effects were also observed when the I -, Br -, and SCN -concentrations were plotted against the NO combination rate constants. Interestingly, the trough concentrations obtained from the bell-shaped curves are comparable to normal plasma levels of halides and pseudohalides, suggesting the potential relevance of these molecules in modulating MPO function. The second-order rate constant of NO binding in the presence of plasma levels of I -, Br -, and SCN -is 1-2-fold lower compared to that obtained in the absence of these molecules and remains unaltered through the Cl -plasma level. When Cl -exceeded the plasma level, the NO combination rate becomes indistinguishable from the second phase of the bell-shaped curve that was obtained in the absence of halides. Our results are consistent with two halide binding sites that could be populated by two halides in which both display distinct effects on the MPO heme iron microenvironment. Myeloperoxidase (MPO) 1 is an abundant heme-containing protein found in neutrophil granules, monocytes, and selected tissue macrophages (1-3). MPO plays an important role in generating an array of toxic oxidants important to host defense (1-3). The molecular mass of the enzyme is 150-165 kDa and the enzyme is comprised of two identical subunits joined by a single disulfide bridge (2). Each subunit consists of a light chain and a heavy chain derived from a single gene product (4). The heavy chains contain an iron bound to a novel protoporphyrin IX derivative that is covalently attached to the heavy chain polypeptide (5, 6). The heme prosthetic groups are approximately 50 Å apart, and a variety of observations suggest that both are functionally identical (7-10). They presumably operate independently in the oxidation of Cl -and in the bactericidal activity of the enzyme (7). Structural studies of both canine and human MPO demonstrate that the heme of MPO is positioned at the base of a deep and narrow cleft and is axially coordinated to the protein through His933 (7-10). The imidazole ring of His95 is located 5.7 Å from the heme iron, while the guanidinium group of Arg239 and the side chain of Gln91 are close to the heme surface and have minimum interatomic distances from the iron atom of 7.0 and 4.5 Å, respectively (7-10). The location of these residues above the heme iron is consistent with the heme iron being the site where hydrogen peroxide •-, nitric oxide (NO), and ascorbic aci

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 (Å) 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 Å. 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