Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme.

We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PR(S17)), selected to represent high resistance by machine learning on genotype-phenotype data. Three of five mutations in PR(S17) correlating with major drug resistance, M46L, G48V, and V82S, and five of 11 natural variations differ from the mutations in two clinically derived extreme mutants, PR20 and PR22 bearing 19 and 22 mutations, respectively. PR(S17), which forms a stable dimer (<10 nM), is ∼10- and 2-fold less efficient in processing the Gag polyprotein than the wild type and PR20, respectively, but maintains the same cleavage order. Isolation of a model precursor of PR(S17) flanked by the 56-amino acid transframe region (TFP-p6pol) at its N-terminus, which is impossible upon expression of an analogous PR20 precursor, allowed systematic comparison of inhibition of TFP-p6pol-PR(S17) and mature PR(S17). Resistance of PR(S17) to eight protease inhibitors (PIs) relative to PR (Ki) increases by 1.5-5 orders of magnitude from 0.01 to 8.4 µM. Amprenavir, darunavir, atazanavir, and lopinavir, the most effective of the eight PIs, inhibit precursor autoprocessing at the p6pol/PR site with IC50 values ranging from ∼7.5 to 60 µM. Thus, this process, crucial for stable dimer formation, shows inhibition ∼200-800-fold weaker than that of the mature PR(S17). TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC50 = 15 µM); amprenavir shows a 2-fold increase in IC50 (∼15 µM), and atazanavir and lopinavir show increased IC50 values of >42 and >70 µM, respectively.

Mutations Proximal to Sites of Autoproteolysis and the α-Helix That Co-evolve under Drug Pressure Modulate the Autoprocessing and Vitality of HIV-1 Protease.

N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67, and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10-35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR(PR20-3) because of the cumulative effect of eight noncluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function.

Exploring selection and recruitment processes for newly qualified nurses: a sequential-explanatory mixed-method study.

AIM: To map current selection and recruitment processes for newly qualified nurses and to explore the advantages and limitations of current selection and recruitment processes. BACKGROUND: The need to improve current selection and recruitment practices for newly qualified nurses is highlighted in health policy internationally. DESIGN: A cross-sectional, sequential-explanatory mixed-method design with 4 components: (1) Literature review of selection and recruitment of newly qualified nurses; and (2) Literature review of a public sector professions' selection and recruitment processes; (3) Survey mapping existing selection and recruitment processes for newly qualified nurses; and (4) Qualitative study about recruiters' selection and recruitment processes. METHODS: Literature searches on the selection and recruitment of newly qualified candidates in teaching and nursing (2005-2013) were conducted. Cross-sectional, mixed-method data were collected from thirty-one (n = 31) individuals in health providers in London who had responsibility for the selection and recruitment of newly qualified nurses using a survey instrument. Of these providers who took part, six (n = 6) purposively selected to be interviewed qualitatively. RESULTS: Issues of supply and demand in the workforce, rather than selection and recruitment tools, predominated in the literature reviews. Examples of tools to measure values, attitudes and skills were found in the nursing literature. The mapping exercise found that providers used many selection and recruitment tools, some providers combined tools to streamline process and assure quality of candidates. CONCLUSIONS: Most providers had processes which addressed the issue of quality in the selection and recruitment of newly qualified nurses. The 'assessment centre model', which providers were adopting, allowed for multiple levels of assessment and streamlined recruitment. There is a need to validate the efficacy of the selection tools.

Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Recruiting competent newly qualified nurses in the London region: An exploratory study.

AIM: This paper reports a study commissioned to address concerns that not all newly qualified nurses (NQNs) were perceived to be competent at the point of appointment to their first post. It seeks to understand how competence is interpreted in the context of selection and recruitment, and explore the different expectations and experiences of employing Trusts across the London region. BACKGROUND: Competence is a significant topic in nursing and there is much literature around the concept, what it means and how it relates to behaviours and values with no universally accepted definition. However, there appears to be little evidence about how competence is assessed in practice in the selection and recruitment of NQNs to their first post. METHODS: The study took a three-phase, mixed method approach including a literature review, an electronic survey to map current assessment and selection procedures, and focus groups to identify the competencies perceived essential by senior nurses. FINDINGS: Most Trusts reported assessing core competencies, and could report how they do this with respect to literacy and numeracy. Employers could describe what they required from NQNs, and how applicants both met and did not meet expectations. Several personal attributes were considered as important as key competences, but these are not described in the KSF or NMC frameworks, and it is not clear how these are assessed in selection processes. CONCLUSION: There appeared to be a large variation in the number and types of competence assessments being used for recruitment, with little consistency in the detail of the assessments, although broadly similar assessment exercises are used. There appears to be little evidence as to the validity of the measures being used and whether in fact they are measuring the competences that are being sought or considered most important. It would appear that practical skills are more easily assessable, but there is a lack of clarity regarding the assessment of those competences that are considered equally important but appear to be more elusive to assessment such as communication and teamwork. It is also unclear how a number of 'personal qualities' described as essential for NQNs are being assessed at recruitment.

The challenge of change in acute mental health services: measuring staff perceptions of barriers to change and their relationship to job status and satisfaction using a new measure (VOCALISE).

BACKGROUND: Health services are subject to frequent changes, yet there has been insufficient research to address how staff working within these services perceive the climate for implementation. Staff perceptions, particularly of barriers to change, may affect successful implementation and the resultant quality of care. This study measures staff perceptions of barriers to change in acute mental healthcare. We identify whether occupational status and job satisfaction are related to these perceptions, as this might indicate a target for intervention that could aid successful implementation. As there were no available instruments capturing staff perceptions of barriers to change, we created a new measure (VOCALISE) to assess this construct. METHODS: All nursing staff from acute in-patient settings in one large London mental health trust were eligible. Using a participatory method, a nurse researcher interviewed 32 staff to explore perceptions of barriers to change. This generated a measure through thematic analyses and staff feedback (N = 6). Psychometric testing was undertaken according to standard guidelines for measure development (N = 40, 42, 275). Random effects models were used to explore the associations between VOCALISE, occupational status, and job satisfaction (N = 125). RESULTS: VOCALISE was easy to understand and complete, and showed acceptable reliability and validity. The factor analysis revealed three underlying constructs: 'confidence,' 'de-motivation' and 'powerlessness.' Staff with negative perceptions of barriers to change held more junior positions, and had poorer job satisfaction. Qualitatively, nursing assistants expressed a greater sense of organisational unfairness in response to change. CONCLUSIONS: VOCALISE can be used to explore staff perceptions of implementation climate and to assess how staff attitudes shape the successful outcomes of planned changes. Negative perceptions were linked with poor job satisfaction and to those occupying more junior roles, indicating a negative climate for implementation in those groups. Staff from these groups may therefore need special attention prior to implementing changes in mental health settings.

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Extreme multidrug resistant HIV-1 protease with 20 mutations is resistant to novel protease inhibitors with P1'-pyrrolidinone or P2-tris-tetrahydrofuran.

Extreme drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with the clinical inhibitor amprenavir (1) and two potent antiviral investigational inhibitors GRL-02031 (2) and GRL-0519 (3). Clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme, which is consistent with dissociation constants (KL) from isothermal titration calorimetry of 40 nM for 3, 178 nM for amprenavir, and 960 nM for 2. High resolution crystal structures of PR20-inhibitor complexes revealed altered interactions compared with the corresponding wild-type PR complexes in agreement with relative inhibition. Amprenavir lacks interactions due to PR20 mutations in the S2/S2' subsites relative to PR. Inhibitors 2 and 3 lose interactions with Arg8' in PR20 relative to the wild-type enzyme because Arg8' shifts to interact with mutated L10F side chain. Overall, inhibitor 3 compares favorably with darunavir in affinity for PR20 and shows promise for further development.

Equality of employment opportunities for nurses at the point of qualification: an exploratory study.

BACKGROUND: Securing employment after qualification is of utmost importance to newly qualified nurses to consolidate knowledge and skills. The factors that influence success in gaining this first post are not known. OBJECTIVES: The study aimed to describe the first post gained after qualification in terms of setting, nature of employment contract and geographical distribution and explore the relationship between a range of factors (including ethnicity) and employment at the point of qualification. DESIGN: An exploratory study using structured questionnaires and secondary analysis of data routinely collected by the universities about students and their progress during their course. SETTINGS: The study was conducted in eight universities within a large, multicultural city in the UK as part of the 'Readiness for Work' research programme. PARTICIPANTS: Eight hundred and four newly qualified nurses who had successfully completed a diploma or degree from one of the universities; a response rate of 77% representing 49% of all graduating students in the study population. METHODS: Data were collected by self-completed semi-structured questionnaires administered to students at the time of qualification and at three months post-qualification. Routinely collected data from the universities were also collected. RESULTS: Fifty two percent of participants had been offered a job at the point of qualification (85% of those who had applied and been interviewed). Of these, 99% had been offered a nursing post, 88% in the city studied, 67% in the healthcare setting where they had completed a course placement. 44% felt "confident" and 32% "very confident" about their employment prospects. Predictors of employment success included ethnicity, specialty of nursing and university attended. Predictors of confidence and preparedness for job seeking included ethnicity, nursing specialty, gender and grade of degree. Newly qualified nurses from non-White/British ethnic groups were less likely to get a job and feel confident about and prepared for job seeking. CONCLUSIONS: This study has demonstrated that ethnicity does lead to employment disadvantage for newly qualified nurses. This is an important contribution towards recognizing and describing the evidence so that appropriate responses and interventions can be developed. It is important that universities and healthcare institutions work closely together to support students at this important time in their nursing career.

Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

Mechanism of dissociative inhibition of HIV protease and its autoprocessing from a precursor.

Dimerization is indispensible for release of the human immunodeficiency virus protease (PR) from its precursor (Gag-Pol) and ensuing mature-like catalytic activity that is crucial for virus maturation. We show that a single-chain Fv fragment (scFv) of a previously reported monoclonal antibody (mAb1696), which recognizes the N-terminus of PR, dissociates a dimeric mature D25N PR mutant with an enhanced dimer dissociation constant (K(d)) in the sub-micromolar range to form predominantly a monomer-scFv complex at a 1:1 ratio, along with small (5-10%) amounts of a dimer-scFv complex. Enzyme kinetics indicate a mixed mechanism of inhibition of the wild-type PR, which exhibits a K(d)<10nM, with effects both on K(m) and k(cat) at an scFv-to-PR ratio of 10:1. ScFv binds to the N-terminal peptide P(1)QITLW(6) of PR and to PR monomers with dissociation constants of ≤30 nM and ~100 nM, respectively. Consistent with an ~400-fold increase in the dissociation of the antibody (K(Ab)) on even addition of an acetyl group to P(1) of the peptide, the antibody fails to inhibit N-terminal autoprocessing of the PR from a model precursor (at ~5 µM). However, subsequent to this cleavage, it sequesters the PR, thus blocking autoprocessing at its C-terminus. A second monoclonal antibody [PRM1 (human monoclonal antibody to PR)], which recognizes part of the flap region (residues 41-47) of the mature PR and its precursor, does not inhibit autoprocessing and ensuing catalytic activity. However, its failure to recognize drug-resistant clinical mutants of PR may be beneficial to monitor the selection of mutations in this region under drug pressure.

HIV-1 protease with 20 mutations exhibits extreme resistance to clinical inhibitors through coordinated structural rearrangements.

The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35-37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2' pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.

Terminal interface conformations modulate dimer stability prior to amino terminal autoprocessing of HIV-1 protease.

The HIV-1 protease (PR) mediates its own release (autoprocessing) from the polyprotein precursor, Gag-Pol, flanked by the transframe region (TFR) and reverse transcriptase at its N- and C-termini, respectively. Autoprocessing at the N-terminus of PR mediates stable dimer formation essential for catalytic activity, leading to the formation of infectious virus. An antiparallel ß-sheet interface formed by the four N- and C-terminal residues of each subunit is important for dimer stability. Here, we present the first high-resolution crystal structures of model protease precursor-clinical inhibitor (PI darunavir or saquinavir) complexes, revealing varying conformations of the N-terminal flanking (S(-4)FNF(-1)) and interface residues (P(1)QIT(4)). A 180° rotation of the T(4)-L(5) peptide bond is accompanied by a new Q(2)-L(5) hydrogen bond and complete disengagement of PQIT from the ß-sheet dimer interface, which may be a feature for intramolecular autoprocessing. This result is consistent with drastically lower thermal stability by 14-20 °C of PI complexes of precursors and the mature PR lacking its PQIT residues (by 18.3 °C). Similar to the TFR-PR precursor, this deletion also results in a darunavir dissociation constant (2 × 10(4))-fold higher and a markedly increased dimer dissociation constant relative to the mature PR. The terminal ß-sheet perturbations of the dimeric structure likely account for the drastically poorer inhibition of autoprocessing of TFR-PR relative to the mature PR, even though significant differences in active site-PI interactions in these structures were not observed. The novel conformations of the dimer interface may be exploited to target selectively the protease precursor prior to its N-terminal cleavage.

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors.

Self-cleavage at the N terminus of HIV-1 protease from the Gag-Pol precursor (autoprocessing) is crucial for stabilizing the protease dimer required for onset of mature-like catalytic activity, viral maturation, and propagation. Among nine clinical protease inhibitors (PIs), darunavir and saquinavir were the most effective in inhibiting wild-type HIV-1 group M precursor autoprocessing, with an IC(50) value of 1-2 µM, 3-5 orders of magnitude higher than their binding affinities to the corresponding mature protease. Accordingly, both group M and N precursor-PI complexes exhibit T(m)s 17-21 °C lower than those of the corresponding mature protease-PI complexes suggestive of markedly reduced stabilities of the precursor dimer-PI ensembles. Autoprocessing of group N (natural variant) and three group M precursors bearing 11-20 mutations associated with multidrug resistance was either weakly responsive or fully unresponsive to inhibitors at concentrations up to a practical limit of approximately 150 µM PI. This observation parallels decreases of up to 8 × 10(3)-fold (e.g., 5 pM to 40 nM) in the binding affinity of darunavir and saquinavir to mature multidrug resistant proteases relative to wild type, suggesting that inhibition of some of these mutant precursors will occur only in the high µM to mM range in extreme PI-resistance, which is an effect arising from coordinated multiple mutations. An extremely darunavir-resistant mutant precursor is more responsive to inhibition by saquinavir. These findings raise the questions whether clinical failure of PI therapy is related to lack of inhibition of autoprocessing and whether specific inhibitors can be designed with low-nM affinity to target autoprocessing.

The L76V drug resistance mutation decreases the dimer stability and rate of autoprocessing of HIV-1 protease by reducing internal hydrophobic contacts.

The mature HIV-1 protease (PR) bearing the L76V drug resistance mutation (PR(L76V)) is significantly less stable, with a >7-fold higher dimer dissociation constant (K(d)) of 71 ± 24 nM and twice the sensitivity to urea denaturation (UC(50) = 0.85 M) relative to those of PR. Differential scanning calorimetry showed decreases in T(m) of 12 °C for PR(L76V) in the absence of inhibitors and 5-7 °C in the presence of inhibitors darunavir (DRV), saquinavir (SQV), and lopinavir (LPV), relative to that of PR. Isothermal titration calorimetry gave a ligand dissociation constant of 0.8 nM for DRV, ∼160-fold higher than that of PR, consistent with DRV resistance. Crystal structures of PR(L76V) in complexes with DRV and SQV were determined at resolutions of 1.45-1.46 Å. Compared to the corresponding PR complexes, the mutated Val76 lacks hydrophobic interactions with Asp30, Lys45, Ile47, and Thr74 and exhibits closer interactions with Val32 and Val56. The bound DRV lacks one hydrogen bond with the main chain of Asp30 in PR(L76V) relative to PR, possibly accounting for the resistance to DRV. SQV shows slightly improved polar interactions with PR(L76V) compared to those with PR. Although the L76V mutation significantly slows the N-terminal autoprocessing of the precursor TFR-PR(L76V) to give rise to the mature PR(L76V), the coselected M46I mutation counteracts the effect by enhancing this rate but renders the TFR-PR(M46I/L76V) precursor less responsive to inhibition by 6 µM LPV while preserving inhibition by SQV and DRV. The correlation of lowered stability, higher K(d), and impaired autoprocessing with reduced internal hydrophobic contacts suggests a novel molecular mechanism for drug resistance.

Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV-1 protease: relevance to drug resistance.

The mature protease from Group N human immunodeficiency virus Type 1 (HIV-1) (PR1(N)) differs in 20 amino acids from the extensively studied Group M protease (PR1(M)) at positions corresponding to minor drug-resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1(N) with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1(M), but with a slightly larger inhibitor-binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor-binding residues 29-32. However, kinetic parameters of PR1(N) closely resemble those of PR1(M), and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug-specific major DRMs. A miniprecursor (TFR 1-61-PR1(N)) comprising the transframe region (TFR) fused to the N-terminus of PR1(N) undergoes autocatalytic cleavage at the TFR/PR1(N) site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (∼6 µM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1-61/PR1(N) cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1(N) relative to PR1(M) include its suitability for column fractionation by size under native conditions and >10-fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.

Revealing the dimer dissociation and existence of a folded monomer of the mature HIV-2 protease.

Purification and in vitro protein-folding schemes were developed to produce monodisperse samples of the mature wild-type HIV-2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2(E37K) (elevated K(d) approximately 20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a T(m) of 60.5 for PR2 as compared with 65.7 degrees C for HIV-1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the T(m) of PR2 increases by 14.8 degrees C in the presence of DRV as compared with 22.4 degrees C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2(T26A)) or a deletion of the C-terminal residues 96-99 (PR2(1-95)), drastically increase the K(d) (>10(5)-fold). PR2(T26A) and PR2(1-95) consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size-exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild-type PR2 and its active-site mutant PR2(D25N) are folded dimers. Addition of twofold excess active-site inhibitor promotes dimerization of PR2(T26A) but not of PR2(1-95), indicating that subunit interactions involving the C-terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.

Modulation of human immunodeficiency virus type 1 protease autoprocessing by charge properties of surface residue 69.

Mature, fully active human immunodeficiency virus protease (PR) is liberated from the Gag-Pol precursor via regulated autoprocessing. A chimeric protease precursor, glutathione S-transferase-transframe region (TFR)-PR-FLAG, also undergoes N-terminal autocatalytic maturation when it is expressed in Escherichia coli. Mutation of the surface residue H69 to glutamic acid, but not to several neutral or basic amino acids, impedes protease autoprocessing in bacteria and mammalian cells. Only a fraction of mature PR with an H69E mutation (PR(H69E)) folds into active enzymes, and it does so with an apparent Kd (dissociation constant) significantly higher than that of the wild-type protease, corroborating the marked retardation of the in vitro N-terminal autocatalytic processing of TFR-PR(H69E) and suggesting a folding defect in the precursor.

Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin.

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.

3'-Intercalation of a N2-dG 1R-trans-anti-benzo[c]phenanthrene DNA adduct in an iterated (CG)3 repeat.

The conformation of the 1 R,2 S,3 R,4 S-benzo[ c]phenanthrene- N (2)-dG adduct, arising from trans opening of the (+)-1 S,2 R,3 R,4 S- anti-benzo[ c]phenanthrene diol epoxide, was examined in 5'- d(ATCGC XCGGCATG)-3'.5'-d(CATGCCG CGCGAT)-3', where X = 1 R,2 S,3 R,4 S-B[ c]P- N (2)-dG. This duplex, derived from the hisD3052 frameshift tester strain of Salmonella typhimurium, contains a (CG) 3 iterated repeat, a hotspot for frameshift mutagenesis. NMR experiments showed a disconnection in sequential NOE connectivity between X (4) and C (5), and in the complementary strand, they showed another disconnection between G (18) and C (19). In the imino region of the (1)H NMR spectrum, a resonance was observed at the adducted base pair X (4) x C (19). The X (4) N1H and G (18) N1H resonances shifted upfield as compared to the other guanine imino proton resonances. NOEs were observed between X (4) N1H and C (19) N (4)H and between C (5) N (4)H and G (18) N1H, indicating that base pairs X (4) x C (19) and C (5) x G (18) maintained Watson-Crick hydrogen bonding. No NOE connectivity was observed between X (4) and G (18) in the imino region of the spectrum. Chemical shift perturbations of greater than 0.1 ppm were localized at nucleotides X (4) and C (5) in the modified strand and G (18) and C (19) in the complementary strand. A total of 13 NOEs between the protons of the 1 R-B[ c]Ph moiety and the DNA were observed between B[ c]Ph and major groove aromatic or amine protons at base pairs X (4) x C (19) and 3'-neighbor C (5) x G (18). Structural refinement was achieved using molecular dynamics calculations restrained by interproton distances and torsion angle restraints obtained from NMR data. The B[ c]Ph moiety intercalated on the 3'-face of the X (4) x C (19) base pair such that the terminal ring of 1 R-B[ c]Ph threaded the duplex and faced into the major groove. The torsion angle alpha' [X (4)]-N3-C2-N2-B[ c]Ph]-C1 was calculated to be -177 degrees, maintaining an orientation in which the X (4) exocyclic amine remained in plane with the purine. The torsion angle beta' [X (4)]-C2-N2-[B[ c]Ph]-C1-C2 was calculated to be 75 degrees. This value governed the 3'-orientation of the B[ c]Ph moiety with respect to X (4). The helical rise between base pairs X (4) x C (19) and C (5) x G (18) increased and resulted in unwinding of the right-handed helix. The aromatic rings of the B[ c]Ph moiety were below the Watson-Crick hydrogen-bonding face of the modified base pair X (4) x C (19). The B[c]Ph moiety was stacked above nucleotide G (18), in the complementary strand.