Juraj Sedláček

Structural Basis of HIV-1 and HIV-2 Protease Inhibition by a Monoclonal Antibody

BACKGROUND Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.

Inhibitor binding at the protein interface in crystals of a HIV-1 protease complex.

Depending on the excess of ligand used for complex formation, the HIV-1 protease complexed with a novel phenylnorstatine inhibitor forms crystals of either hexagonal (P6(1)) or orthorhombic (P2(1)2(1)2(1)) symmetry. The orthorhombic form shows an unusual complexity of crystal packing: in addition to one inhibitor molecule that is bound to the enzyme active site, the second inhibitor molecule is bound as an outer ligand at the protein interface. Binding of the outer ligand apparently increases the crystal-quality parameters so that the diffraction data allow solution of the structure of the complex at 1.03 A, the best resolution reported to date. The outer ligand interacts with all four surrounding HIV-1 protease molecules and has a bent conformation owing to its accommodation in the intermolecular space. The parameters of the solved structures of the orthorhombic and hexagonal forms are compared.

Stabilization of antibody structure upon association to a human carbonic anhydrase IX epitope studied by X-ray crystallography, microcalorimetry, and molecular dynamics simulations.

Specific antibodies interfere with the function of human tumor‐associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan‐like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR‐H1 and CDR‐H3. In contrast, the overall conformations and positions of CDR‐H2 and CDR‐L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR‐H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation‐libration‐screw (TLS) method shows that librations of the entire heavy chain variable domain (VH) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR‐H3. Moreover, it was observed that one proton is transferred from the environment to the protein‐ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM‐GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer. Proteins 2008.

Potent inhibition of drug-resistant HIV protease variants by monoclonal antibodies.

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.

A distinct binding mode of a hydroxyethylamine isostere inhibitor of HIV-1 protease

Crystallization conditions for an HIV-1 protease-inhibitor complex were optimized to produce crystals suitable for X-ray diffraction experiments. The X-ray structure of the HIV-1 protease complex was solved and refined at 3.1 A resolution. In contrast to Saquinavir, the mimetic hydroxy group of the inhibitor Boc-Phe-Psi[(S)-CH(OH)CH(2)NH]-Phe-Glu-Phe-NH(2) is placed asymmetrically with respect to the non-crystallographic twofold axis of the protease dimer so that hydrogen bonds between the amino group of the inhibitor and the catalytic aspartates can be formed. The inhibitor binds in the centre of the active site by a compact network of hydrogen bonds to Gly27, Gly127, Asp25, Asp125 and via the buried water molecule W301 to Ile50 and Ile150.

Structure of a single‐chain Fv fragment of an antibody that inhibits the HIV‐1 and HIV‐2 proteases

The monoclonal antibody 1696, which was raised against the HIV-1 protease, inhibits the catalytic activity of the enzyme from both the HIV-1 and HIV-2 strains. The antibody cross-reacts with peptides containing the N-terminus of the enzyme, which is highly conserved between these strains. The crystal structure of a single-chain Fv fragment of 1696 (scFv-1696) in the non-complexed form, solved at 1.7 A resolution, is compared with the previously reported non-complexed Fab-1696 and antigen-bound scFv-1696 structures. Large conformational changes in the third hypervariable region of the heavy chain and differences in relative orientation of the variable domains are observed between the different structures.

Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinant Escherichia coli.

Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of thetac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 °C, respectively. The differential rates of synthesis of prochymosin inclusion, and, for comparison, of β-lactamase and β-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth. At 28 °C the induced expression of prochymosin gene was almost blocked. Addition of novobiocin did not alleviate this effect. In fact, it strengthtened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level. At 40 °C the differential rate of prochymosin synthesis was markedly enhanced. Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause. The stimulation of cloned heterologous gene expression at 40 °C and inhibition at 28 °C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.

Regular arrangement of periodates bound to lysozyme.

The structure of tetragonal hen egg-white lysozyme soaked in a periodate solution has been determined to a resolution of 1.8 A. Four high-occupancy periodate positions have been identified on the basis of the anomalous signal of the I atoms. The four periodates exhibit a regular rectangular arrangement on the surface of the lysozyme molecule. No similar regular arrangement was found either in lysozyme crystals soaked in other heavy-atom anions or in other structures from the Protein Data Bank. Depending on their position on the surface of the protein, the periodate ions deviate to a varying extent from ideal octahedral geometry.

Processing, Purification, and Kinetic Characterization of the Gag-Pol Encoded Retroviral Proteinase of Myeloblastosis Associated Virus Expressed in E. Coli

Gag-Pol frameshift translational products of avian retroviruses (e. g. myeloblastosis associated virus, MAV, or Rous sarcoma virus, RSV) contain a putative proteinase species that maps between the NC/PR and PR/RT processing sites. Such Gag-Pol proteinase deffers from the gag-encoded proteinase (Gag PR, 124 amino acids length) by a carboxy-terminal extension of 7 amino acids (numbered 125 to 131), i. e. -IGRATVL (see Refs. 1,2,3, for review). While the main species of the processing proteinase of avian retroviruses, Gag PR (known also as pl5gag) was characterized to considerable details (4, 5, 6, 7), direct description of avian retroviral Gag-Pol proteinases remained very limited (3) and substantial knowledge was missing. We attempted to analyze in detail recombinant gag-pol encoded species of the MAV avian retroviral proteinase (identical to that of RSV, except for a conservative substitution at amino acid residue 84, Ref. 8) using a truncated, E. coli expressed in-frame precursor that retains the natural NC/PR cleavage site and is translationally terminated at the position of the PR/RT site. This work showed that under conditions favourable for processing, an unexpected cleavage occurs between amino acid residues 126 and 127, and that this cleavage bears characteristics of a regular self-processing step. The cleavage product, termed PR(+IleGly), represents an enzyme with catalytic parameters not much different but distinguishable from those of the gag-encoded proteinase.