Flap Region Research Articles

Recent Turbulence Model Advances Applied to Multielement Airfoil Computations

A one-equation linear turbulence model and two-equation nonlinear explicit algebraic stress model (EASM) are applied to the flow over a multielement airfoil. The effect of the K-epsilon and K-omega forms of the two-equation model are explored, and the K-epsilon form is shown to be deficient in the wall-bounded regions of adverse pressure gradient flows. A new K-omega form of EASM is introduced. Nonlinear terms present in EASM are shown to improve predictions of turbulent shear stress behind the trailing edge of the main element and near midflap. Curvature corrections are applied to both the one- and two-equation turbulence models and yield only relatively small local differences in the flap region, where the flow field undergoes the greatest curvature. Predictions of maximum lifts are essentially unaffected by the turbulence model variations studied.

Interactions of a Novel Inhibitor from an ExtremophilicBacillus sp. with HIV-1 Protease: IMPLICATIONS FOR THE MECHANISM OF INACTIVATION

The active site cleft of the HIV-1 protease (PR) is bound by two identical conformationally mobile loops known as flaps, which are important for substrate binding and catalysis. The present article reports, for the first time, an HIV-1 PR inhibitor, ATBI, from an extremophilic Bacillus sp. The inhibitor is found to be a hydrophilic peptide with Mr of 1147, and an amino acid sequence of Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu. Sequence homology exhibited no similarity with the reported peptidic inhibitors of HIV-1 PR. Investigation of the kinetics of the enzyme-inhibitor interactions revealed that ATBI is a noncompetitive and tight binding inhibitor with the IC(50) and K(i) values 18.0 and 17.8 nm, respectively. The binding of the inhibitor with the enzyme and the subsequent induction of the localized conformational changes in the flap region of the HIV-1 PR were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-42 residues, which are present at the proximity of the flaps. We have demonstrated by fluorescence and circular dichroism studies that ATBI binds in the active site of the HIV-1 PR and thereby leads to the inactivation of the enzyme. Based on our results, we propose that the inactivation is due to the reorganization of the flaps impairing its flexibility leading toward inaccessibility of the substrate to the active site of the enzyme.

Tonotopic and Somatotopic Representation in the Nucleus basalis of the Barn Owl, Tyto alba

We have investigated the somatosensory and auditory representations in the nucleus basalis of the barn owl. In pigeons and finches, the nucleus basalis contains a representation of the beak and an auditory area. In the barn owl, the nucleus basalis also contains a complete somatotopic map of the head and body (as in the budgerigar), with a tonotopically organized auditory area in close proximity to the representation of the facial ruff and the preaural area. Recordings within and around the nucleus basalis revealed predominantly (about 80%) contralateral responses to somatic stimulation. The somatotopic map was oriented with the head down and rostral. Penetrations revealed an over-representation of the feet in dorsal basalis, followed by the rest of the body and wings more ventrally. Towards more rostral positions in nucleus basalis, responses from the head and beak predominated ventrally. The auditory response area was encountered below the region that responded to stimulation of the facial ruff and preaural flap regions and above a region responsive to beak stimulation. Auditory responses were tonotopically organized, with low best frequencies dorsal. Some penetrations yielded predominantly monaural responses with a fairly broad dynamic range, similar to those recorded from the ventral nucleus of the lateral lemniscus (LLV) and the cochlear nucleus angularis, whereas other penetrations contained predominantly binaural responses sensitive to interaural time differences (ITD). The physiological responses could be predicted on the basis of auditory projections to the nucleus basalis. An injection of biotinylated dextran amine (BDA) in the auditory region of nucleus basalis retrogradely labeled cells in both the caudal and rostral parts of the intermediate lateral lemniscal nucleus (LLIc and LLIr), and a few cells in the anterior part of the dorsal lateral lemniscal nucleus (LLDa, previously known as nucleus ventralis lemnisci lateralis, pars anterior, or VLVa) and in the posterior part of the dorsal lateral lemniscal nucleus (LLDp, previously known as nucleus ventralis lemnisci lateralis, pars posterior, or VLVp). A large injection of cholera toxin B-chain (CTB) into the nucleus basalis also produced dense retrograde labeling of a previously unidentified nucleus on the lateral aspect of the rostral pons, that we here call nucleus pontis externus (PE). An injection of CTB into PE produced dense retrograde labeling of the contralateral dorsal column nuclei and anterograde labeling of the ipsilateral lateral and dorsolateral nucleus basalis. Together these results define major somatosensory and auditory projections to the owl telencephalon that bypass the thalamus.

Dynamics of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor.

A significant determinant for the broad substrate specificity of the metallo-beta-lactamases from Bacteroides fragilis and other similar organisms is the presence of a plastic substrate binding site that is nevertheless capable of tight substrate binding in the Michaelis complex. To achieve these two competing ends, the molecule apparently employs a flexible flap that closes over the active site in the presence of substrate. These characteristics imply that dynamic changes are an important component of the mechanism of action of these enzymes. The backbone and tryptophan side chain dynamics of the metallo-beta-lactamase from B. fragilis have been examined using (15)N NMR relaxation measurements. Two states of the protein were examined, in the presence and absence of a tight-binding inhibitor. Relaxation measurements were analyzed by the model-free method. Overall, the metallo-beta-lactamase molecule is rigid and shows little flexibility except in loops. The flexibility of the loop that covers the active site is not unusually great as compared to the other loops of the protein. Local motion on a picosecond time scale was found to be very similar throughout the protein in the presence and absence of the inhibitor, but a significant difference was observed in the motions on a nanosecond time scale (tau(e)). Large-amplitude motions with a time constant of about 1.3 ns were observed for the flexible flap region (residues 45-55) in the absence of the inhibitor. These motions were completely damped out in the presence of the inhibitor. In addition, the motion of a tryptophan side chain at the tip of the beta-hairpin of the flap shows a very significant difference in motion on the ps time scale. These results indicate that the motions of the polypeptide chain in the flap region can be invoked to explain both the wide substrate specificity (the free form has considerable amplitude of motion in this region) and the catalytic efficiency of the metallo-beta-lactamase (the motions are damped out when the inhibitor and by implication a substrate binds in the active site).

Fine characterization of the antigenic site within the flap region in the protease protein of HIV-1.

The aspartate protease encoded by human immunodeficiency virus type 1 is essential for cleavage of the gag and gag-pol precursor proteins. The majority of HIV-1-antibody-positive sera react with the protease. In this study we used a substitution set of peptides for detailed characterization of the earlier defined antigenic site (aa 44-58) within the central "flap" region, also important in the context of conformational flexibility during protease inhibitor binding. We found that isoleucine at position 54 was important for creating an antigenic site required for binding of anti-HIV-1 sera. The identification of structurally essential amino acids in the flap region of HIV-1 PR may have important implications in future development of antiviral drugs.

Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease.

The Q7K/L331/L631 HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with k(q1) = 6.85 x 10(9) M(-1) s(-1) and k(q2) = 1.88 x 10(9) M(-1) s(-1), that merge into one in the complex with k(q) = 1.78 x 10(9) M(-1) s(-1). 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

Complex craters: Relationship of stratigraphy and rings to impact conditions

One of the key issues associated with the understanding of large scale impacts is how the observable complex crater structural features (e.g., central peaks and pits, flat floors, ring shaped ridges and depressions, stratigraphic modifications, and faults) relate to the impactor's parameters (e.g., radius, velocity, and density) and the nonobservable transient crater measures (e.g., depth of penetration and diameter at maximum penetration). We have numerically modeled large‐scale impacts on planets for a range of impactor parameters, gravity and planetary material strengths. From these we found that the collapse of the transient cavity results in the development of a tall, transient central peak that oscillates and drives surface waves that are arrested by the balance between gravitational forces and planetary strength to produce a wide range of the observed surface features. In addition, we found that the underlying stratigraphy is inverted outside of the transient cavity diameter (overturned flap region), but not inside. This change in stratigraphy is observable by remote sensing, drilling, seismic imaging and gravity mapping techniques. We used the above results to develop scaling laws and to make estimates of the impact parameters for the Chicxulub impact and also compared the calculated stratigraphic profile with the internal structure model developed by Hildebrand et. al. [1998], using gravity, seismic and other field data. For a stratigraphy rotation diameter of 90 km, the maximum depth of penetration is ∼43 km. The impactor diameter was also calculated. From the scaling relationships we get for a 2.7 g/cm3 asteroid impacting at 20 km/s, or a 1.0 g/cm3 comet impacting at 40 km/s, an impactor diameter of ∼13 km, and for a comet impacting at 60 km/s, an impactor diameter of ∼10 km.

4-Hydroxy-5,6-Dihydro-2 H-pyran-2-ones. 3. Bicyclic and hetero-aromatic ring systems as 3-position scaffolds to bind to S 1′ and S 2′ of the HIV-1 protease enzyme

5,6-Dihydro-2 H-pyran-2-ones are potent inhibitors of HIV-1 protease, which bind to the S 1, S 2, S 1′, and S 2′ pockets and have a unique binding mode with the catalytic aspartyl groups and the flap region of the enzyme. Efforts to explore 3-position heterocyclic scaffolds that bind to the S 1′ and S 2′ pockets have provided a number of selected analogs that display high HIV-1 protease inhibitory activity.

FKBP-type peptidyl-prolyl cis-trans isomerase from a sulfur-dependent hyperthermophilic archaeon, Thermococcus sp. KS-1.

A gene encoding an FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) was cloned from a hyperthermophilic archaeon, Thermococcus sp. KS-1, and sequenced. This gene encoded an FKBP with 159 amino-acid residues with a molecular mass of 17.6kDa. Two insertion sequences with 13 and 44 amino acids were found in the regions corresponding to the bulge and flap regions of human FKBP-12, respectively. Comparison with other archaeal FKBP sequences obtained from reported genome sequences revealed that the insertion sequences in the bulge and flap regions were common to archaeal FKBPs. It was also revealed that archaeal FKBPs are classified into two groups: one is approx. 17kDa and the other 27kDa. This Thermococcus FKBP (TcFK) belonged to the smaller archaeal FKBP. In this TcFK, 9 out of 15 amino acid residues forming the FK506 binding pocket of human FKBP12 were found. This gene was expressed in Escherichia coli and the recombinant protein was purified. The purified protein showed PPIase activity and its activity was inhibited by FK506 with an IC50 of 7 microM. This enzyme showed high kinetic stability with a half-life of 40 min at 100 degrees C. Catalytic efficiency of this recombinant PPIase was 1.2-times higher with the substrate N-succinyl-A-L-P-F-p-nitroanilide than with N-succinyl-A-A-P-F-p-nitroanilide.

A cooperative folding unit in HIV-1 protease. Implications for protein stability and occurrence of drug-induced mutations.

We investigated the HIV-1 protease molecule for the occurrence of cooperative folding units, i.e. structural units that exhibit a relatively stronger protection against unfolding than do other parts of the molecule. Calculated unfolding penalties are used to delineate folding units. This procedure identifies a folding core in HIV-1 protease, based on an ensemble of denatured states derived from native structures, comprising a spatially close unit of residues 84-91, 74-78 and 22-32, the last of which contains the active site residues D25, T26 and G27. Observed enzyme mutations of HIV-1 protease, either naturally occurring or induced by drug therapy, are found in regions that are not structurally designed to withstand unfolding. These mutations are especially likely to occur in the flap region, a part of the protein which is not essential for the stability of the protein, but does contribute significantly to the stability of protease-drug complexes. A similar avoidance of structurally protected regions in the reverse transcriptase enzyme is also observed.

The structural stability of the HIV-1 protease

The most common strategy in the development of HIV-1 protease inhibitors has been the design of high affinity transition state analogs that effectively compete with natural substrates for the active site. A second approach has been the development of compounds that inactivate the protease by destabilizing its quaternary or tertiary structure. A successful optimization of these strategies requires an accurate knowledge of the energetics of structural stabilization and binding, and the identification of those regions in the protease molecule that are critical to stability and function. Here the energetics of stabilization of the HIV-1 protease has been measured for the first time by high sensitivity differential scanning calorimetry. These studies have permitted the evaluation of the different components of the Gibbs energy of stabilization (the enthalpy, entropy and heat capacity changes). The stability of the protease is pH-dependent and due to its dimeric nature is also concentration-dependent. At pH 3.4 the Gibbs energy of stabilization is close to 10 kcal/mol at 25°C, consistent with a dissociation constant of 5 × 10−8 M. The stability of the protease increases at higher pH values. At pH 5, the Gibbs energy of stabilization is 14.5 kcal/mol at 25°C, consistent with a dissociation constant of 2.3 × 10−11 M. The pH dependence of the Gibbs energy of stabilization indicates that between pH 3.4 and pH 5 an average of 3–4 ionizable groups per dimer become protonated upon unfolding. A structure-based thermodynamic analysis of the protease molecule indicates that most of the Gibbs energy of stabilization is provided by the dimerization interface and that the isolated subunits are intrinsically unstable. The Gibbs energy, however, is not uniformly distributed along the dimerization interface. The dimer interface is characterized by the presence of clusters of residues (hot spots) that contribute significantly and other regions that contribute very little to subunit association. At the dimerization interface, residues located at the carboxy and amino termini contribute close to 75% of the total Gibbs energy (Cys95, Thr96, Leu97, Asn98 and Phe99 and Pro1, Ile3, Leu5). Residues Thr26, Gly27 and Asp29 located at the base of the active site are also important, and to a lesser extent Gly49, Ile50, Gly51 located at the tip of the flap region. The structure-based thermodynamic analysis also predicts the existence of regions of the protease with only marginal stability and a high propensity to undergo independent local unfolding. In particular, the flap region occupies a very shallow energy minimum and its conformation can easily be affected by relatively small perturbations. This property of the protease can be related to the ability of some mutations to elicit resistance towards certain inhibitors.

Structural Role of the 30's Loop in Determining the Ligand Specificity of the Human Immunodeficiency Virus Protease,

The structural basis of ligand specificity in human immunodeficiency virus (HIV) protease has been investigated by determining the crystal structures of three chimeric HIV proteases complexed with SB203386, a tripeptide analogue inhibitor. The chimeras are constructed by substituting amino acid residues in the HIV type 1 (HIV-1) protease sequence with the corresponding residues from HIV type 2 (HIV-2) in the region spanning residues 31-37 and in the active site cavity. SB203386 is a potent inhibitor of HIV-1 protease (Ki = 18 nM) but has a decreased affinity for HIV-2 protease (Ki = 1280 nM). Crystallographic analysis reveals that substitution of residues 31-37 (30's loop) with those of HIV-2 protease renders the chimera similar to HIV-2 protease in both the inhibitor binding affinity and mode of binding (two inhibitor molecules per protease dimer). However, further substitution of active site residues 47 and 82 has a compensatory effect which restores the HIV-1-like inhibitor binding mode (one inhibitor molecule in the center of the protease active site) and partially restores the affinity. Comparison of the three chimeric protease structures with those of HIV-1 and SIV proteases complexed with the same inhibitor reveals structural changes in the flap regions and the 80's loops, as well as changes in the dimensions of the active site cavity. The study provides structural evidence of the role of the 30's loop in conferring inhibitor specificity in HIV proteases.

Molecular basis of resistance to HIV-1 protease inhibition: a plausible hypothesis.

The binding thermodynamics of the HIV-1 protease inhibitor acetyl pepstatin and the substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln, corresponding to one of the cleavage sites in the gag, gag-pol polyproteins, have been measured by direct microcalorimetric analysis. The results indicate that the binding of the peptide substrate or peptide inhibitor is entropically driven; i.e., it is characterized by an unfavorable enthalpy and a favorable entropy change, in agreement with a structure-based thermodynamic analysis based upon an empirical parameterization of the energetics. Dissection of the binding enthalpy indicates that the intrinsic interactions are favorable and that the unfavorable enthalpy originates from the energy cost of rearranging the flap region in the protease molecule. In addition, the binding is coupled to a negative heat capacity change. The dominant binding force is the increase in solvent entropy that accompanies the burial of a significant hydrophobic surface. Comparison of the binding energetics obtained for the substrate with that obtained for synthetic nonpeptide inhibitors indicates that the major difference is in the magnitude of the conformational entropy change. In solution, the peptide substrate has a higher flexibility than the synthetic inhibitors and therefore suffers a higher conformational entropy loss upon binding. This higher entropy loss accounts for the lower binding affinity of the substrate. On the other hand, due to its higher flexibility, the peptide substrate is more amenable to adapt to backbone rearrangements or subtle conformational changes induced by mutations in the protease. The synthetic inhibitors are less flexible, and their capacity to adapt is more restricted. The expected result is a more pronounced effect of mutations on the binding affinity of the synthetic inhibitors. On the basis of the thermodynamic differences in the mode of binding of substrate and synthetic inhibitors, it appears that a key factor to understanding resistance is given by the relative balance of the different forces that contribute to the binding free energy and, in particular, the balance between conformational and solvation entropy.

Structure of a G48H mutant of HIV-1 protease explains how glycine-48 replacements produce mutants resistant to inhibitor drugs

The crystal structure of human immunodeficiency virus type 1 (HIV-1) protease mutant G48H with peptidic inhibitor U-89360E is described. Comparison with wild-type protease-inhibitor complex shows that mutation of flap residue 48 to histidine allows stabilizing van der Waals contacts between the side chains of His 48 and Phe 53 as well as between His 48 and the P 2′ and P 3′ inhibitor subsites. The flap region is less mobile than in the wild-type enzyme. A model of saquinavir-resistant mutant protease G48V in complex with saquinavir predicts interactions similar to those found in the G48H crystal. Energetic calculations confirm the similarity of the His 48 and Val 48 interactions.

A fast estimate of electrostatic group contributions to the free energy of protein-inhibitor binding.

Dissecting ligand-protein binding free energies in individual contributions of protein residues (which are referred to here as 'group contributions') is of significant importance. For example, such contributions could help in estimating the corresponding mutational effects and in studies of drug resistance problems. However, the meaning of group contributions is not always uniquely defined and the approximations for rapid estimates of such contributions are not well developed. In this paper, the nature of group contributions to binding free energy is examined, focusing particularly on electrostatic contributions which are expected to be well behaved. This analysis examines different definitions of group contributions; the 'relaxed' group contributions that represent the change in binding energy upon mutation of the given residue to glycine, and the 'non-relaxed' group contributions that represent the scaled Coulomb interaction between the given residue and the ligand. Both contributions are defined and evaluated by the linear response approximation (LRA) of the PDLD/ S method. The present analysis considers the binding of pepstatin to endothiapepsin and 23 of its mutants as a test case for a neutral ligand. The 'non-relaxed' group contributions of 15 endothiapepsin residues show significant peaks in the 'electrostatic fingerprint'. The residues that contribute to the electrostatic fingerprint are located in the binding site of endothiapepsin. They include the aspartic dyad (Asp32, Asp215) with adjacent residues and the flap region. Twelve of these 15 residues have a heavy atom distance of <3.75 A to pepstatin. The contributions of 8 (10) of these 12 residues can be reconciled with the calculated 'relaxed' group contributions where one allows the protein and solvent (solvent only) to relax upon mutation of the given residue to glycine. On the other hand, it was found that residues at the second 'solvation shell' can have relaxed contributions that are not captured by the non-relaxed approach. Hence, whereas residues with significant non-relaxed electrostatic contributions are likely to contribute to binding, residues with small non-relaxed contributions may still affect the binding energy. At any rate, it is established here that even in the case of uncharged inhibitors it is possible to use the non-relaxed electrostatic fingerprint to detect 'hot' residues that are responsible for binding. This is significant since some versions of the non-relaxed approximation are faster by several orders of magnitude than more rigorous approaches. The general applicability of this approach is outlined, emphasizing its potential in studies of drug resistance where it is crucial to have a rapid way of anticipating the effect of mutation on both drug binding and catalysis.

Anatomy of the postauricular island "revolving door" flap ("flip-flop" flap).

Reconstruction following resection of auricular (cavum conchae) lesions may be done with a retroauricular rotation flap. Recently there has been revived interest in this elegant reconstructive procedure. Although the vascular anatomy of the area was studied, no direct study of flap anatomy was reported. Six fresh adult male cadaveric dissections of the retroauricular area were performed. The skin and underlying subcutaneous tissue layer were reflected to correspond with flap size, and anatomic structures were studied. Dissection was carried out on 12 ears. The origin of the occipital belly of the occipitofrontalis muscle arising from the posterior mastoid region was identified in four patients and only as part of the fascial layer overlying the posterior mastoid region. Only a small portion of the sternocleidomastoid tendon at best is possibly incorporated in the flap. It seems that only a negligible contribution to the flap is derived from the temporalis muscle. The posterior auricular muscle was identified in all patients and its origin from the skull was (in all patients) included or bordered the posterior flap region. The posterior auricular artery (PAA) was seen in all 12 dissections. The artery was adjacent to the styloid process medial to the parotid gland superficially between the auricular cartilage and the mastoid process. The PAA was then found on the periosteum of the mastoid process, ascending deep to the posterior auricular muscle. The flap seems to be a truly fasciocutaneous flap with small, questionable, superior and anteroinferior muscular contributions, and an inclusion of the rather small posterior auricular muscle. As reported in other studies, blood supply to the area seems to be derived from the PAA.

Role of adipocytes in the muscle tissue of Atlantic salmon ( Salmo salar ) in the uptake, release and retention of water-soluble fraction of crude oil hydrocarbons

The uptake and depuration of the water-sol‐uble fraction (WSF) of hydrocarbons of crude petroleum by Atlantic salmon (Salmosalar) has previously been examined in terms of whole muscle. The hypothesis that the tainting WSF in the muscle was retained primarily by adipocytes has been investigated by the isolation of adipocytes and the subsequent analysis for hydrocarbons in adipocytes. After 96 h exposure of market-sized Atlantic salmon to 0.2 ppm WSF, adipocytes isolated from the belly flap region of the muscle tissue accumulated 14.3 times more WSF (59.4 ppm) than the dorsal white muscle (4.2 ppm), while 54% of the tainting WSF in the dorsal white muscle was found to be stored in associated adipocytes. When returned to clean seawater, WSF accumulated in the dorsal white muscle was released much faster than that in the adipocytes. These results indicated that the loose association of WSF with the nonlipid portion of white muscle, mainly muscle cells and intercellular fluid, is responsible for the rapid discharge of WSF from the dorsal muscle tissue in the early stages of depuration. After 4 d of depuration, the adipocytes became the principal storage site of residual WSF in white muscle and the depuration of WSF from muscle tissue then reflected the release of WSF from adipocytes in the muscle tissue. After 20 d of depuration, 10.7 ppm of tainting WSF in the form of high molecular weight aromatic hydrocarbons (mainly C4-benzenes, naphthalene and alkylated naphthalenes) were still present in adipocytes, while in the dorsal white muscle only a trace of total WSF was detected. Increases in the number of aromatic rings and the alkylations on the rings enhanced the accumulation and retention of individual hydrocarbons in both adipocytes and white muscle. From these studies we conclude that it is the adipocytes in the muscle tissue which control the actual accumulation and release of hydrocarbons in the whole muscle tissue of Atlantic salmon.

Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit.

The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) [Cheng Y.-S. E., Yin, F.H., Foundling, S., Blomstrom, D. & Kettner, C. A. (1990) Proc. Natl Acad. Sci. USA 87, 9660-9664] and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.

Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants

Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants

Resistance-related mutations in the HIV-1 protease gene of patients treated for 1 year with the protease inhibitor ritonavir (ABT-538).

To define genotypic and phenotypic resistance patterns following prolonged therapy with the protease inhibitor ritonavir (ABT-538). Seven HIV-1-infected patients, all but one previously treated with dideoxynucleoside analogues (zidovudine, didanosine, zalcitabine), were treated for 1 year with ritonavir. Direct solid-phase sequencing of the protease gene starting from plasma derived viral RNA followed by comparison to phenotypic drug resistance data. The most frequent amino-acid substitutions occurring upon administration of the protease inhibitor were V82A/F (substrate binding site), I54V (flap region), A71V and L10I. Additional mutations found in more than one patient were I15V, M36I, I84V and I93L. Mutation L63P was found both in pre- and post-ritonavir samples. Phenotypic drug resistance assays confirmed resistance to ritonavir in post-treatment samples (approximately 170-fold) and showed cross-resistance to indinavir (approximately 30-fold) and partially to saquinavir (approximately fivefold). At 1 year of treatment, one patient without known resistance-associated mutations in the protease gene still showed a substantial rise in CD4 cell count accompanied by a more than 2.4 log decrease in RNA viral load. However, at week 78, mutations R8Q, E34K, R57K, L63P and I84V were detected and the treatment benefit was partially lost. Long-term treatment with ritonavir is associated with the emergence of multiple mutations in the HIV-1 protease gene. The mutations L10I, I54V, L63P, A71V, V82A/F and I84V correspond to known drug-resistance mutations for ritonavir and other protease inhibitors. Phenotypic resistance to ritonavir was detected in a majority of ritonavir-treated patients at 1 year of treatment. In addition, long-term ritonavir treatment selects for cross-resistance to the protease inhibitors indinavir and saquinavir. This argues against sequential therapy with several protease inhibitors. Delayed resistance in one patient was accompanied with a prolonged increase in CD4 cell count and decrease in viral load suggesting a temporary benefit of treatment.