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Understanding the catalytic mechanism of SARS-CoV-2 main protease (Mpro) can help in guiding drug design of specific small-molecule antivirals. A 2.0 Å resolution room-temperature X-ray crystal structure of inactive C145A mutant Mpro in complex with the octapeptide Ac-SAVLQSGF-CONH2 that resembles a Michaelis complex is reported., SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145–His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3′ by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.

HIV-1 protease is indispensable for virus propagation and an important therapeutic target for antiviral inhibitors to treat AIDS. As such inhibitors are transition-state mimics, a detailed understanding of the enzyme mechanism is crucial for the development of better anti-HIV drugs. Here, we used room-temperature joint X-ray/neutron crystallography to directly visualize hydrogen atoms and map hydrogen bonding interactions in a protease complex with peptidomimetic inhibitor KVS-1 containing a reactive nonhydrolyzable ketomethylene isostere, which, upon reacting with the catalytic water molecule, is converted into a tetrahedral intermediate state, KVS-1TI. We unambiguously determined that the resulting tetrahedral intermediate is an oxyanion, rather than the gem-diol, and both catalytic aspartic acid residues are protonated. The oxyanion tetrahedral intermediate appears to be unstable, even though the negative charge on the oxyanion is delocalized through a strong n → π* hyperconjugative interaction into the nearby peptidic carbonyl group of the inhibitor. To better understand the influence of the ketomethylene isostere as a protease inhibitor, we have also examined the protease structure and binding affinity with keto-darunavir (keto-DRV), which similar to KVS-1 includes the ketomethylene isostere. We show that keto-DRV is a significantly less potent protease inhibitor than DRV. These findings shed light on the reaction mechanism of peptide hydrolysis catalyzed by HIV-1 protease and provide valuable insights into further improvements in the design of protease inhibitors.

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA–SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA–SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15 N and 13 C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15 N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15 N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N–H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA–SP1 cleavage.

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure-activity relationship (SAR) study of noncovalent compounds that bind in the enzyme's substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor's P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag–Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag–Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions., NMR studies define how the P182L mutation alters intramolecular interactions to increase HSP27 accessibility for numerous binding proteins, compromising its chaperone function in patient cell‐derived motor neurons.

Following excision from the Gag-Pol polyprotein, HIV-1 reverse transcriptase is released as an asymmetric homodimer comprising two p66 subunits that are structurally dissimilar but identical in amino acid sequence. Subsequent cleavage of the RNase H domain from only one of the subunits, denoted p66', results in the formation of the mature p66/p51 enzyme in which catalytic activity resides in the p66 subunit, and the p51 subunit (derived from p66') provides a supporting structural scaffold. Here, we probe the interaction of the p66/p66' asymmetric reverse transcriptase precursor with HIV-1 protease by pulsed Q-band double electron-electron resonance EPR spectroscopy to measure distances between nitroxide labels introduced at surface-engineered cysteine residues. The data suggest that the flexible, exposed linker between the RNaseH and connection domains in the open state of the p66' subunit binds to the active site of protease in a configuration that is similar to that of extended peptide substrates.

Significance The root cause of pathology in sickle cell disease is the polymerization of the mutant hemoglobin S upon deoxygenation in the tissues to form fibers. Both the amount of fiber at equilibrium and the kinetics of fiber formation depend on the partial pressure of oxygen. We show that control of polymerization by oxygen at equilibrium can be better explained by a recent extension of the famous two-state allosteric model of Monod, Wyman, and Changeux. Because of the unusual kinetics, polymerization is far out of equilibrium, which explains why patients with the disease manage to survive, while those with high levels of fetal hemoglobin and those with sickle trait (the heterozygous condition) have relatively benign conditions., The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D2O with hydrogenous glycerol to maximize deuterium incorporation into the protein, with no detrimental effects on the growth of quality crystals suitable for neutron diffraction experiments.

The modern energy landscape theory of protein folding predicts multiple folding pathways connecting a myriad of unfolded conformations and a well-defined folded state. However, direct experimental observation of heterogeneous folding pathways is difficult. Naturally evolved proteins typically exhibit a smooth folding energy landscape for fast and efficient folding by avoiding unfavorable kinetic traps. In this case, rapid fluctuations between unfolded conformations result in apparent two-state behavior and make different pathways indistinguishable. However, the landscape roughness can be different, depending on the selection pressures during evolution. Here, we characterize the unusually rugged folding energy landscape of human immunodeficiency virus-1 protease monomer using single-molecule Forster resonance energy transfer spectroscopy. Our data show that fluctuations between unfolded conformations are slow, which enables the experimental observation of heterogeneous folding pathways as predicted by the landscape theory. Although the landscape ruggedness is sensitive to the mutations and fluorophore locations, the folding rate is similar for various protease constructs. The natural evolution of the protease to have a rugged energy landscape likely results from intrinsic pressures to maintain robust folding when human immunodeficiency virus-1 mutates frequently, which is essential for its survival.

An extremely drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with two potent antiviral investigational inhibitors. GRL-5010A and GRL-4410A were designed to introduce hydrogen bond interactions with the flexible flaps of the PR by incorporating gem-difluorines and alkoxy, respectively, at the C4 position of the bis-THF of darunavir. PR20 provides an excellent model for high level resistance, since clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme. GRL-5010A and GRL-4410A show inhibition constants of 4.3 ± 7.0 and 1.7 ± 1.8 nM, respectively, for PR20, compared to the binding affinity of 41 ± 1 nM measured for darunavir. Crystal structures of PR20 in complexes with the two inhibitors confirmed the new hydrogen bond interactions with Gly 48 in the flap of the enzyme. The two new compounds are more effective than darunavir in inhibiting mature PR20 and show promise for further development of antiviral agents targeting highly resistant PR mutants.

The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr−Purcell−Meiboom−Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease−Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti-HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.

Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.

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 of42 and70 μM, respectively.

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 analog 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 analog relative to wild type enzyme. 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.

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 β-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 β-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 β-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.

We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNA CUA pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p -benzoylphenylalanine ( p BzF). The most potent peptide (G12: GIXVSL; X = p BzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the p BzF residue with the ϵ-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.

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 A 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.

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 PR2E37K (elevated Kd ∼20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a Tm of 60.5 for PR2 as compared with 65.7°C for HIV-1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the Tm of PR2 increases by 14.8°C in the presence of DRV as compared with 22.4°C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2T26A) or a deletion of the C-terminal residues 96–99 (PR21–95), drastically increase the Kd (>105-fold). PR2T26A and PR21–95 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 PR2D25N are folded dimers. Addition of twofold excess active-site inhibitor promotes dimerization of PR2T26A but not of PR21–95, 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.

Previously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct N(CCG)-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the N(CCG)-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtype B and C HIV-1 reference panels. The parental Fab 3674 is 10-20-fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format ( approximately 50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format ( approximately 120 kDa) across the subtype B and C reference panels.

No drug has been targeted specifically for HIV-2 (human immunodeficiency virus type 2) infection despite its increasing prevalence worldwide. The antiviral HIV-1 (human immunodeficiency virus type 1) protease (PR) inhibitor darunavir and the chemically related GRL98065 and GRL06579A were designed with the same chemical scaffold and different substituents at P2 and P2′ to optimize polar interactions for HIV-1 PR (PR1). These inhibitors are also effective antiviral agents for HIV-2-infected cells. Therefore, crystal structures of HIV-2 PR (PR2) complexes with the three inhibitors have been solved at 1.2-A resolution to analyze the molecular basis for their antiviral potency. Unusually, the crystals were grown in imidazole and zinc acetate buffer, which formed interactions with the PR2 and the inhibitors. Overall, the structures were very similar to the corresponding inhibitor complexes of PR1 with an RMSD of 1.1 A on main-chain atoms. Most hydrogen-bond and weaker C–H…O interactions with inhibitors were conserved in the PR2 and PR1 complexes, except for small changes in interactions with water or disordered side chains. Small differences were observed in the hydrophobic contacts for the darunavir complexes, in agreement with relative inhibition of the two PRs. These near-atomic-resolution crystal structures verify the inhibitor potency for PR1 and PR2 and will provide the basis for the development of antiviral inhibitors targeting PR2.

The HIV-1 protease enzyme is indispensable for viral maturation, making it a major potential target of anti-HIV therapy. Its role is to split newly formed Gag and Gag-Pol polyproteins to produced finished structural and functional proteins — itself included. The early events in the autoprocessing of HIV-1 protease, in which a precursor dimer is thought to undergo intramolecular cleavage, have now been visualized using NMR spectroscopy and paramagnetic relaxation enhancement. This reveals that although primarily monomeric, the protease is also present as transient encounter complexes that occupy a wide range of orientations relative to the mature dimer. The N-terminal region makes transient intra- and intersubunit contacts with the substrate binding site, allowing autocleavage to occur when the correct dimer orientation is sampled by the encounter complex. HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins. The mature protease is only active as a dimer, with catalytic residues contributed by each subunit. The precursor of the active protease undergoes 'maturation' via the intramolecular cleavage of a dimeric species, but it is not clear how this cleavage reaction occurs. The early events in N-terminal auto-processing were visualized using NMR spectroscopy, and it was determined that the precursor forms transient, lowly populated dimeric encounter complexes that occupy a wide range of orientations relative to the mature dimer. The N-terminal region makes transient intra- and intersubunit contacts with the substrate binding site, enabling auto-cleavage to occur when the correct dimer orientation is sampled by the encounter complex ensemble. HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation1,2. The mature protease is active only as a dimer3,4,5 with each subunit contributing catalytic residues6. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer5,7,8,9,10,11, concomitant with the appearance of mature-like catalytic activity7,9. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit β-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor5,12. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species13,14,15,16, we show that the mini-precursor forms highly transient, lowly populated (3–5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.

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.

Following CD4 receptor binding to the HIV-1 envelope spike (Env), the conserved N-heptad repeat (NHR) region of gp41 forms a coiled-coil that is a precursor to the fusion reaction. Although it has been a target of drug and vaccine design, there are few monoclonal antibody (mAb) tools with which to probe the antigenicity and immunogenicity specifically of the NHR coiled-coil. Here, we have rescued HIV-1-neutralizing anti-NHR mAbs from immune phage display libraries that were prepared (i) from b9 rabbits immunized with a previously described mimetic of the NHR coiled-coil, N35CCG-N13, and (ii) from an HIV-1 infected individual. We describe a rabbit single-chain Fv fragment (scFv), 8K8, and a human Fab, DN9, which specifically recognize NHR coiled-coils that are unoccupied by peptide corresponding to the C-heptad repeat or CHR region of gp41 (e.g. C34). The epitopes of 8K8 and DN9 were found to partially overlap with that of a previously described anti-NHR mAb, IgG D5; however, 8K8 and DN9 were much more specific than D5 for unoccupied NHR trimers. The mAbs, including a whole IgG 8K8 molecule, neutralized primary HIV-1 of clades B and C in a pseudotyped virus assay with comparable, albeit relatively modest potency. Finally, a human Fab T3 and a rabbit serum (both non-neutralizing) were able to block binding of D5 and 8K8 to a gp41 NHR mimetic, respectively, but not the neutralizing activity of these mAbs. We conclude from these results that NHR coiled-coil analogs of HIV-1 gp41 elicit many Abs during natural infection and through immunization, but that due to limited accessibility to the corresponding region on fusogenic gp41 few can neutralize. Caution is therefore required in targeting the NHR for vaccine design. Nevertheless, the mAb panel may be useful as tools for elucidating access restrictions to the NHR of gp41 and in designing potential improvements to mimetics of receptor-activated Env.

An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR(RE) containing Asp25Arg and Gly49Glu mutations, and PR(RER) containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. (1)H-(15)N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR(RE) has a high tendency to aggregate, PR(RER) exists mainly as a folded monomer at 25-35 microM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR(RE) sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR(RER) sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR(RER) protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1.

Internal motion in proteins fulfills a multitude of roles in biological processes. NMR spectroscopy has been applied to elucidate protein dynamics at the atomic level, albeit at a low resolution, and is often complemented by molecular dynamics simulation. However, it is critical to justify the consistency between simulation results and conclusions often drawn from multiple experiments in which uncertainties arising from assumed motional models may not be explicitly evaluated. To understand the role of the flaps of HIV-1 protease dimer in substrate recognition and protease function, many molecular dynamics simulations have been performed. The simulations have resulted in various proposed models of the flap dynamics, some of which are more consistent than others with our working model previously derived from experiments. However, using the working model to discriminate among the simulation results is not straightforward because the working model was derived from a combination of NMR experiments and crystal structure data. In this study, we use the NMR chemical shifts and relaxation data of the protease “monomer” rather than structural data to narrow down the possible conformations of the flaps of the “dimer”. For the first time, we show that the tips of the flaps in the unliganded protease dimer interact with each other in solution. Accordingly, we discuss the consistency of the simulations with the model derived from all experimental data. Proteins 2008. © 2007 Wiley-Liss, Inc.

An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of approximately 20 micoM. Under these conditions, NMR spectra show that the mature protease lacking its terminal beta-sheet residues 1-4 and 96-99 (PR(5-95)) exhibits a stable monomer fold spanning the region 10-90 that is similar to that of the single subunit of the wild-type dimer and the dimer bearing a D25N mutation (PR(D25N)). Urea-induced unfolding monitored both by changes in (1)H-(15)N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR(5-95) monomer displays a transition profile similar to that of the PR(D25N) dimer (50% unfolded (U(50)) = approximately 1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ((SFNF)PR(D25N)) decreases the stability of the fold (U(50) = approximately 1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR(T26A) (U(50) = 2.5 M) and (SFNF)PR(D25N) monomers and compared with PR(D25N/T26A) monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of (SFNF)PR(D25N) that may relate to the increase in the equilibrium dissociation constant (K(d)) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.

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.

The flexibility of the glycine-rich flaps are known to be essential for the catalytic activity of the HIV-1 Protease but their exact conformations at the different stages of the enzymatic pathway remain yet to be determined. While hundreds of crystal structures of protease-inhibitor complexes have been solved, only a dozen of unbound protease structures have been reported to date. These structures reveal a large heterogeneity of flap conformations, ranging from closed, to semi-open and wide-open conformations. We used here NMR spectroscopy to determine the flap orientation of the unbound protease in solution. The conformation of the flaps was determined by comparing the measured residual dipolar couplings to reference crystal structures representing the closed, semi-open and wide-open states. The data clearly indicate that the apo protease adopts a closed conformation in solution, similar to an inhibitor-bound state.We also compared the effect of high-pressure on the flap conformations of an apo and inhibitor-bound protease by measuring backbone residual dipolar couplings up to 1 kbar. A rigid-body motion model was derived to characterize the pressure-induced conformational changes of the flaps. While a minimal rearrangement of the flaps was observed in the case of the inhibitor-protease complex, we observed a significant pressure-induced rotation of the flaps in the case of the apo protease. These results highlight the intrinsic conformational flexibility of the unbound protease and offer a new perspective to understand the mechanism of ligand and inhibitor binding.

Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR(F53L) showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR(F53L) was determined in the unliganded form at 1.35 Angstrom resolution in space group P4(1)2(1)2. The tips of the flaps in PR(F53L) had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50'. The changes in interactions between the flaps agreed with the reduced stability of PR(F53L) relative to wild-type PR. The altered flap interactions in the unliganded form of PR(F53L) suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.

Flexibility of the glycine-rich flaps is known to be essential for catalytic activity of the HIV-1 protease, but their exact conformations at the different stages of the enzymatic pathway remain subject to much debate. Although hundreds of crystal structures of protease-inhibitor complexes have been solved, only about a dozen inhibitor-free protease structures have been reported. These latter structures reveal a large diversity of flap conformations, ranging from closed to semi-open to wide open. To evaluate the average structure in solution, we measured residual dipolar couplings (RDCs) and compared these to values calculated for crystal structures representative of the closed, semi-open, and wide-open states. The RDC data clearly indicate that the inhibitor-free protease, on average, adopts a closed conformation in solution that is very similar to the inhibitor-bound state. By contrast, a highly drug-resistant protease mutant, PR20, adopts the wide-open flap conformation.

HIV-1 envelope glycoprotein-mediated fusion is driven by the concerted coalescence of the HIV-1 gp41 N- and C-helical regions, which results in the formation of 6-helix bundles. These two regions are considered prime targets for peptides and antibodies that inhibit HIV-1 entry. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by monitoring the temporal sequence of conformational states of HIV-1 gp41 during the course of HIV-1-mediated cell-cell fusion by quantitative video microscopy using reagents that bind to N- and C-helical regions, respectively. Env-expressing cells were primed by incubation with target cells at different times at 37 degrees C followed by washing. The reactivity of triggered gp41 to the NC-1 monoclonal antibody, which we demonstrate here to bind to N-helical gp41 trimers, increased rapidly to a maximal level in the primed state but decreased once stable fusion junctions had formed. In contrast, reactivity with 5-helix, which binds to the C-helical region of gp41, increased continuously as a function of time following the priming. The peptide N36(Mut(e,g)) reduced NC-1 monoclonal antibody binding and enhanced 5-helix binding, consistent with the notion that this molecule promotes dissociation of gp41 trimers. This inactivation pathway may be important for the design of entry inhibitors and vaccine candidates.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein flanked by Gag sequences (r-preNC) was expressed in Escherichia coli and purified. HIV-1 proteinase cleaved r-preNC to the "mature" NCp7 form, which is comprised of 55 residues. Further incubation resulted in cleavages of NCp7 itself between Phe16 and Asn17 of the proximal zinc finger domain and between Cys49 and Thr50 in the C-terminal part. Kinetic parameters determined for the cleavage of oligopeptides corresponding to the cleavage sites in r-preNC correlated well with the sequential processing of r-preNC. Mutations of Asn17 were introduced to alter the susceptibility of NC protein to HIV-1 proteinase. While mutating Asn17 to Ala resulted in a protein which was processed in a manner similar to that of the wild type, mutating it to Phe or Leu resulted in proteins which were processed at a substantially higher rate at this site than the wild type. Mutation of Asn17 to Lys or Gly resulted in proteins which were very poorly cleaved at this site. Oligopeptides containing the same amino acid substitutions at the cleavage site of the proximal zinc finger domain were also tested as substrates of the proteinase, and the kinetic parameters agreed well with the semiquantitative results obtained with the protein substrates.

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.

Interactions between the C-terminal interface residues (96-99) of the mature HIV-1 protease were shown to be essential for dimerization, whereas the N-terminal residues () and Arg(87) contribute to dimer stability (Ishima, R., Ghirlando, R., Tozser, J., Gronenborn, A. M., Torchia, D. A., and Louis, J. M. (2001) J. Biol. Chem. 276, 49110-49116). Here we show that the intramonomer interaction between the side chains of Asp(29) and Arg(87) influences dimerization significantly more than the intermonomer interaction between Asp(29) and Arg(8'). Several mutants, including T26A, destablize the dimer, exhibit a monomer fold, and are prone to aggregation. To alleviate this undesirable property, we designed proteins in which the N- and C-terminal regions can be linked intramolecularly by disulfide bonds. In particular, cysteine residues were introduced at positions 2 and 97 or 98. A procedure for the efficient preparation of intrachain-linked polypeptides is presented, and it is demonstrated that the Q2C/L97C variant exhibits a native-like single subunit fold. It is anticipated that monomeric proteases of this kind will aid in the discovery of novel inhibitors aimed at binding to the monomer at the dimerization interface. This extends the target area of current inhibitors, all of which bind across the active site formed by both subunits in the active dimer.

A widely applicable strategy is presented for efficient and rapid production of small water soluble peptides expressed as fusion proteins with the immunoglobulin-binding domain of streptococcal protein G. A simple extraction and purification scheme that includes a protease cleavage step to release the target peptide is described. The yield of authentic target peptide exceeds 10 mg per liter of culture. Production of U-13C, 15N and highly deuterated U-13C, 15N isotope labeled peptide is demonstrated for the 11 residue S2 peptide, corresponding to the C-terminus of the α-subunit of transducin, and the coiled coil trimerization domain from cartilage matrix protein (CMPcc), respectively. Heteronuclear two-dimensional NMR spectra are used for initial peptide characterization.

We have determined the high-resolution solution structure of the oxidized form of a chimeric human and Escherichia coli thioredoxin (TRX(HE)) by NMR. The overall structure is well-defined with a rms difference for the backbone atoms of 0.27 +/- 0.06 A. The topology of the protein is identical to those of the human and E. coli parent proteins, consisting of a central five-stranded beta-sheet surrounded by four alpha-helices. Analysis of the interfaces between the two domains derived from the human and E. coli sequences reveals that the general hydrophobic packing is unaltered and only subtle changes in the details of side chain interactions are observed. The packing of helix alpha(4) with helix alpha(2) across the hybrid interface is less optimal than in the parent molecules, and electrostatic interactions between polar side chains are missing. In particular, lysine-glutamate salt bridges between residues on helices alpha(2) and alpha(4), which were observed in both human and E. coli proteins, are not present in the chimeric protein. The origin of the known reduced thermodynamic stability of TRX(HE) was probed by mutagenesis on the basis of these structural findings. Two mutants of TRX(HE), S44D and S44E, were created, and their thermal and chemical stabilities were examined. Improved stability toward chaotropic agents was observed for both mutants, but no increase in the denaturation temperature was seen compared to that of TRX(HE). In addition to the structural analysis, the backbone dynamics of TRX(HE) were investigated by (15)N NMR relaxation measurements. Analysis using the model free approach reveals that the protein is fairly rigid with an average S(2) of 0.88. Increased mobility is primarily present in two external loop regions comprising residues 72-74 and 92-94 that contain glycine and proline residues.

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6 HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6 HB trimer and the membrane affinity of gp41's ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41's transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

The molecular basis for high resistance to clinical inhibitors of HIV-1 protease (PR) was examined for the variant designated PRP51 that was selected for resistance to darunavir (DRV). High resolution crystal structures of PRP51 with the active site D25N mutation revealed a ligand-free form and an inhibitor-bound form showing a unique binding site and orientation for DRV. This inactivating mutation is known to increase the dimer dissociation constant and decrease DRV affinity of PR. The PRP51-D25N dimers were in the open conformation with widely separated flaps, as reported for other highly resistant variants. PRP51-D25N dimer bound two DRV molecules and showed larger separation of 8.7 A between the closest atoms of the two flaps compared with 4.4 A for the ligand-free structure of this mutant. The ligand-free structure, however, lacked van der Waals contacts between Ile50 and Pro81' from the other subunit in the dimer, unlike the majority of PR structures. DRV is bound inside the active site cavity; however, the inhibitor is oriented almost perpendicular to its typical position and exhibits only 2 direct hydrogen bond and two water-mediated interactions with atoms of PRP51-D25N compared with 11 hydrogen bond interactions seen for DRV bound in the typical position in wild-type enzyme. The atypical location of DRV may provide opportunities for design of novel inhibitors targeting the open conformation of PR drug-resistant mutants.

Nearly 50 % of the amino acid residues of HIV-1 protease contain methyl side-chains, most of which appear to be organized into two clusters: the inner cluster that nearly surrounds the active site and the outer cluster that contains the hydrophobic core which stabilizes the inhibitor-free protease structure. NMR relaxation experiments sensitive to motions of methyl groups on the sub-nanosecond and the milli-microsecond time-scales revealed flexible methyl groups in residues that link the two clusters, the methyl groups of L10, L23, V75, and L76. We hypothesize that flexibility at the junctions of these clusters allows the protease to minimize conformational changes upon drug-binding. The two-methyl cluster motif appears to be a common structural feature among retroviral proteases and may play a similar role throughout this family of enzymes.

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-A resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wild-type are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D.

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.