From the Cover: Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH₂]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.     

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity. Interestingly, these two NATs use different catalytic strategies to accomplish substrate-specific acetylation. In archaea, where this modification is less prevalent, only one NAT enzyme has been identified. Surprisingly, this enzyme is able to acetylate NatA and NatE substrates and is believed to represent an ancestral NAT variant from which the eukaryotic NAT machinery evolved. To gain insight into the evolution of NAT enzymes, we determined the X-ray crystal structure of an archaeal NAT from Sulfolobus solfataricus (ssNAT). Through the use of mutagenesis and kinetic analysis, we show that the active site of ssNAT represents a hybrid of the NatA and NatE active sites, and we highlight features of this protein that allow it to facilitate catalysis of distinct substrates through different catalytic strategies, which is a unique characteristic of this enzyme. Taken together, the structural and biochemical data presented here have implications for the evolution of eukaryotic NAT enzymes and the substrate specificities therein.The cotranslational process of amino-terminal acetylation occurs on a majority of eukaryotic proteins and mediates many biological processes, including cellular apoptosis, enzymatic regulation, protein localization, and protein degradation (1–4). In humans, three major amino-terminal acetyltransferase (NAT) complexes, called NatA, NatB, and NatC, are responsible for modifying ∼85% of all proteins that undergo amino-terminal acetylation (5). These complexes are conserved in yeast, exist as obligate heterodimers, and are differentiated from one another on the basis of substrate specificity, which is dictated by the amino-terminal sequence of the substrate protein (5, 6). Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6–11). Three additional human NAT enzymes, NatD–NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12–14).The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and human NatE, which is the independently active NAA50 enzyme (15, 16). NatA, which harbors the most diversity for substrate selection, is responsible for modifying the majority of all amino-terminally acetylated proteins, which it accomplishes by recognizing proteins with amino-terminal Ala-, Cys-, Gly-, Ser-, Thr-, or Val- residues (5). NatE/NAA50 has only one known biologically relevant substrate that contains the amino-terminal sequence Met-Leu-Gly-Pro, of which only the amino-terminal Met- is absolutely required for catalysis (14). These structures and their accompanying biochemical characterization have provided significant insight into the mechanisms of substrate specificity and catalysis used by NAT enzymes. Interestingly, although NatA and NatE both catalyze α-amino group acetylation, they use unique catalytic strategies and substantially different substrate amino-terminal residue binding pockets to achieve sequence-based substrate specificity.Although only six amino-terminally acetylated proteins have been identified in Escherichia coli, large-scale proteomics studies of three different archaeal species revealed that ∼14–29% of all proteins isolated are acetylated at their amino terminus (17–20). Analysis of the thermophilic archaea Sulfolobus solfataricus genome resulted in the identification of a protein with 37% sequence identity to human NAA10 that is believed to be the only NAT in this species (21). In agreement with this hypothesis, the same study showed that this protein is able to independently acetylate NatA and NatE substrates, as well as other amino-terminal sequences corresponding to NatB and NatC substrates in vitro (21) Furthermore, no auxiliary subunit homologs could be identified in this genome, suggesting that this archaeal species dedicates just one protein to a task that requires at least nine unique proteins in humans. This ancestral NAT variant, herein referred to as ssNAT, is therefore an ideal candidate for probing the evolution of this family of enzymes and the mechanisms they use to achieve substrate-specific acetylation. Here, we report the X-ray crystal structure of the ssNAT enzyme at 1.98 Å resolution, along with a detailed comparison of ssNAT with the NatA and NatE structures. We combine information from the structure and accompanying alignments to generate a series of ssNAT mutants that we kinetically characterized to dissect the molecular features that promote the acetylation promiscuity observed for this enzyme and to propose evolutionary events that led to a diverse family of substrate-specific monomeric and heterodimeric NATs.     

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease–substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease–substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate residue has distal effects on other residues’ interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease–substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.Resistant pathogens evolve under the selective pressure of drug therapies, commonly by acquiring mutations in the drug target (1–4). Most of these mutations cluster around the drug-binding site and alter key interactions between the drug and its target. Strikingly, mutations in other off-target proteins have also been reported to contribute to drug resistance (5–8) where the mechanism of resistance is not as straightforward to rationalize. In the case of HIV-1, mutations in the target protease gene confer resistance to protease inhibitors (PIs) and correlate with emergence of mutations elsewhere in Gag. There are currently nine protease inhibitors (PIs) that are US Food and Drug Administration (FDA) approved for clinical use including in highly active antiretroviral therapy (HAART) (9), and all are competitive inhibitors binding at the active site.HIV-1 protease is a key antiviral drug target due to its essential function of processing Gag and Gag-Pol polyproteins in viral maturation (10–12). Under the selective pressure of PI-including therapy regimens, viral variants carrying mutations in the protease gene impair the inhibitor efficacy. Although the PIs become weaker binders of these resistant protease variants, the substrates are still hydrolyzed (13, 14), skewing the balance between inhibitor binding and substrate processing in favor of the latter. Earlier work from our group revealed the molecular determinants of this fine balance and formulated the substrate envelope hypothesis to effectively explain the molecular mechanism of resistance due to primary active site mutations (15). Among primary protease mutations, I50V is commonly observed in patients failing therapy with the PIs amprenavir (APV) and darunavir (DRV) (16–18). Residue 50 is located at the flap tip of the flexible loop (50s loop) that controls the access of substrates and competitive inhibitors to the protease active site. In addition to conferring resistance to PIs, the I50V mutation also impairs substrate processing (19). The loss of catalytic efficiency due to I50V is compensated by secondary mutations, in particular A71V (20), which is observed in more than 50% of patient sequences bearing I50V (14).Several mutations in Gag both within cleavage sites and elsewhere coevolve with primary protease mutations contributing to viral fitness and possibly drug resistance (5, 7, 21–25). Particularly, mutations in the p1-p6 cleavage site are statistically associated with I50V protease mutation in the viral sequences retrieved from patients (Fig. 1) (26). The Gag L449F mutation rescues the protease activity by 10-fold, whereas P453L, despite being distal from the catalytic site, causes a 23-fold enhancement (19). However, the molecular basis for the selection advantage of these correlated mutations and the mechanism by which the compensatory mutations restore substrate recognition in drug resistance is not clear. In this study, we report the structural basis for the coevolution of I50V/A71V protease with the p1-p6 substrate. Through a series of cocrystal structures, the Gag mutations L449F and P453L were shown to enhance van der Waals (vdW) interactions between the substrate and mutant protease, whereas R452S results in an additional hydrogen bond. Unexpectedly, the P453L substrate mutation causes a conformational change in the protease flap loop, revealing the molecular mechanism by which this distal substrate mutation is able to enhance substrate–protease interactions. In addition, molecular dynamics simulations suggest that coevolution restores the dynamics at the active site, a key aspect of substrate recognition and turnover that is largely uncharacterized.Open in a separate windowFig. 1.HIV-1 protease and p1-p6 cleavage site coevolution with I50V primary drug resistance mutation. (A) p1-p6 cleavage site sequence and the most common coevolution mutations at P1′, P4′, and P5′ sites. (B) Residues 50 and 71 are indicated as spheres on the homodimeric HIV-1 protease structure. (C) Frequency of mutations in the p1-p6 cleavage site without (dark blue) and with (gray) any mutations at position 50 of the protease. The difference is statistically significant for LP1′, RP4′, and PP5′. Data from ref. 26. (D) Side chains of the substrate residues LP1′, RP4′, and PP5′ and the protease residue I50 are shown as sticks. Monomers of HIV-1 protease are in light purple and green, and the substrate is red in B and D.     

Drug Resistance Mechanism of M46I-Mutation-Induced Saquinavir Resistance in HIV-1 Protease Using Molecular Dynamics Simulation and Binding Energy Calculation

Drug-resistance-associated mutation in essential proteins of the viral life cycle is a major concern in anti-retroviral therapy. M46I, a non-active site mutation in HIV-1 protease has been clinically associated with saquinavir resistance in HIV patients. A 100 ns molecular dynamics (MD) simulation and MM-PBSA calculations were performed to study the molecular mechanism of M46I-mutation-based saquinavir resistance. In order to acquire deeper insight into the drug-resistance mechanism, the flap curling, closed/semi-open/open conformations, and active site compactness were studied. The M46I mutation significantly affects the energetics and conformational stability of HIV-1 protease in terms of RMSD, RMSF, Rg, SASA, and hydrogen formation potential. This mutation significantly decreased van der Waals interaction and binding free energy (∆G) in the M46I–saquinavir complex and induced inward flap curling and a wider opening of the flaps for most of the MD simulation period. The predominant open conformation was reduced, but inward flap curling/active site compactness was increased in the presence of saquinavir in M46I HIV-1 protease. In conclusion, the M46I mutation induced structural dynamics changes that weaken the protease grip on saquinavir without distorting the active site of the protein. The produced information may be utilized for the discovery of inhibitor(s) against drug-resistant HIV-1 protease.     

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I–V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD⁺- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1–NAD⁺ and Ndi1–UQ2 complexes show overlapping binding sites for the NAD⁺ and quinone substrates.     

Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems

Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI toxin/immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn²⁺-dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual β-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.     

Protein protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein

Protein-protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, protein-protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of "hot spots" and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef-SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules.     

Predictors of Current Housing Status Among HIV-Seropositive Injection Drug Users (IDUs): Results from a 1-Year Study

Using longitudinal data collected from 821 HIV-seropositive injection drug users (IDUs) who participated in a multi-site behavioral intervention study, we identified predictors of current housing status at baseline and 12-month follow-up time points. The study was conducted in Baltimore, Miami, New York, and San Francisco from 2001 to 2005. Logistic regression, incorporating the general estimating equations (GEE) method was performed. Multivariate analysis found that Miami participants (OR = 0.56) were less likely to report having current housing (P < 0.05). Among the potential barriers to housing, lower income (OR = 0.68), injection cocaine/crack use (OR = 0.66) and recent incarceration (OR = 0.10) were statistically significant (P < 0.05). Among the potential facilitators of housing, case management (OR = 1.38), outpatient drug treatment attendance (OR = 1.74), and social support (OR = 1.39) were significant. The association between social support and housing was stronger among those who had been recently incarcerated. Additional research is needed to identify types of support and resources beyond what is currently provided in order to better serve housing needs of HIV-seropositive IDUs. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.     

Structural and mechanistic insights into the activation of Stromal interaction molecule 1 (STIM1)

Calcium influx through the Ca(2+) release-activated Ca(2+) (CRAC) channel is an essential process in many types of cells. Upon store depletion, the calcium sensor in the endoplasmic reticulum, STIM1, activates Orai1, a CRAC channel in the plasma membrane. We have determined the structures of SOAR from Homo sapiens (hSOAR), which is part of STIM1 and is capable of constitutively activating Orai1, and the entire coiled coil region of STIM1 from Caenorhabditis elegans (ceSTIM1-CCR) in an inactive state. Our studies reveal that the formation of a SOAR dimer is necessary to activate the Orai1 channel. Mutations that disrupt SOAR dimerization or remove the cluster of positive residues abolish STIM1 activation of Orai1. We identified a possible inhibitory helix within the structure of ceSTIM1-CCR that tightly interacts with SOAR. Functional studies suggest that the inhibitory helix may keep the C-terminus of STIM1 in an inactive state. Our data allowed us to propose a model for STIM1 activation.     

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.