Abstract: The crystal structure of the Midwest Center for Structural Genomics target APC35832, a 14.7-kDa cytosolic protein from Bacillus stearothermophilus, has been determined at 1.3 A resolution by the single anomalous diffraction method from a mercury soaked crystal. The APC35832 protein is a representative of large group of bacterial and archeal proteins entirely consisting of the Toprim (topoisomerase-primase) domain. This domain is found in the catalytic centers of many enzymes catalyzing phosphodiester bond formation or cleavage, but the function of small Toprim domain proteins remains unknown. Consistent with the sequence analysis, the APC35832 structure shows a conserved Toprim fold, with a central 4-stranded parallel beta-sheet surrounded by four alpha-helixes. Comparison of the APC35832 structure with its closest structural homolog, the catalytic core of bacteriophage T7 primase, revealed structural conservation of a metal binding site and isothermal titration calorimetry indicates that APC35832 binds Mg(2+) with a sub-millimolar dissociation constant (K(d)). The APC35832-Mg(2+) complex structure was determined at 1.65 A and reveals the role of conserved acidic residues in Mg(2+) ion coordination. The structural similarities to other Toprim domain containing proteins and potential function and substrates of APC35832 are discussed in this article. Proteins 2008. (c) 2007 Wiley-Liss, Inc.
Abstract: Human immunodeficiency virus (HIV) encodes an aspartic protease (PR) that cleaves viral polyproteins into mature proteins, thus leading to the formation of infectious particles. Protease inhibitors (PIs) are successful virostatics. However, their efficiency is compromised by antiviral resistance. In the PR sequence of viral variants resistant to the PI nelfinavir, the mutations D30N and L90M appear frequently. However, these two mutations are seldom found together in vivo, suggesting that there are two alternative evolutionary pathways leading to nelfinavir resistance. Here we analyze the proteolytic activities, X-ray structures, and thermodynamics of inhibitor binding to HIV-1 PRs harboring the D30N and L90M mutations alone and in combination with other compensatory mutations. Vitality values obtained for recombinant mutant proteases and selected PR inhibitors confirm the crucial role of mutations in positions 30 and 90 for nelfinavir resistance. The combination of the D30N and L90M mutations significantly increases the enzyme vitality in the presence of nelfinavir, without a dramatic decrease in the catalytic efficiency of the recombinant enzyme. Crystal structures, molecular dynamics simulations, and calorimetric data for four mutants (D30N, D30N/A71V, D30N/N88D, and D30N/L90M) were used to augment our kinetic data. Calorimetric analysis revealed that the entropic contribution to the mutant PR/nelfinavir interaction is less favorable than the entropic contribution to the binding of nelfinavir by wild-type PR. This finding is supported by the structural data and simulations; nelfinavir binds most strongly to the wild-type protease, which has the lowest number of protein-ligand hydrogen bonds and whose structure exhibits the greatest degree of fluctuation upon inhibitor binding.
Abstract: 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 (V(H)) 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 2007. (c) 2007 Wiley-Liss, Inc.
Abstract: The crystallization of a given protein is a hard task being even more complicated when the protein shows a hydrophobic behavior. In the case of photosynthetic proteins, the difficulty of the experiments increased due to the high light sensitivity. Aqueous solutions of photosystem II core complex (OEC PSII) of Pisum sativum were screened for crystallization conditions using standard crystallization methods. Crystal improvement was achieved by counter-diffusion technique in single capillaries of 0.2 mm inner diameter with a three-layer configuration. The use of this advanced crystallization technique-for the first time applied to the crystallization of membrane proteins-improves the reproducibility of the experiments allowing the initial crystal characterization, and facilitates the manipulation under light protection.
Abstract: The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.
Abstract: HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.
Abstract: 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.
Abstract: The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH(2) has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K(i) values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
Abstract: 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.
Abstract: 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.
Abstract: The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.
Abstract: 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.
Abstract: We describe a 5-year-old boy with a unique congenital cataract caused by deposition of numerous birefringent, pleiochroic and macroscopically prismatic crystals. Crystal analysis with subsequent automatic Edman degradation and matrix-associated laser desorption ionization time-of-flight mass spectrometry have identified the crystal-forming protein as gammaD-crystallin (CRYGD) lacking the N-terminal methionine. Sequencing of the CRYGD gene has shown a heterozygous C-->A transversion in position 109 of the inferred cDNA (36R-->S transversion of the processed, N-terminal methionine-lacking CRYGD). The lens protein crystals were X-ray diffracting, and our crystal structure solution at 2.25 A suggests that mutant R36S CRYGD has an unaltered protein fold. In contrast, the observed crystal packing is possible only with the mutant protein molecules that lack the bulky Arg36 side chain. This is the first described case of human cataract caused by crystallization of a protein in the lens. It involves the third known mutation in the CRYGD gene but offers, for the first time, a causative explanation of the phenotype.
Abstract: The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.
