Using Multiconformation Continuum Electrostatics to Compare Chloride Binding Motifs in α-Amylase, Human Serum Albumin, and Omp32

Ions are a ubiquitous component of the cellular environment, transferring into cells through membrane-embedded proteins. Ions bind to proteins to regulate their charge and function. Here, using multiconformation continuum electrostatics (MCCE), we show that the changes of chloride binding to α-amylase, human serum albumin (HSA) and Omp32 with pH, and of α-amylase with mutation agree well with experimental data. The three proteins represent three different types of binding. In α-amylase, chloride is bound in a specific buried site. Chloride binding is strongly coupled to the protonation state of a nearby lysine. MCCE calculates an 11-fold change in chloride affinity between the wild-type α-amylase and the K300R mutant, in good agreement with the measured 10-fold change. Without considering the coupled protonation reaction, the calculated affinity change would be more than 10⁶-fold. In HSA, chlorides are distributed on the protein surface. Although HSA has a negative net charge, it binds more anions than cations. There are no highly occupied binding sites in HSA. Rather, there are many partially occupied sites near clusters of basic residues. The relative affinity of bound ions of different charges is shown to depend on the distribution of charged residues on the surface rather than the overall net charge of the protein. The calculated strong pH dependence of the number of chlorides bound and the anion selectivity agree with those of previous experiments. In Omp32, chlorides are stabilized in an anion-selective transmembrane channel in a pH-independent manner. The positive electrostatic potential in Omp32 results in about two chlorides and no cations bound in the transmembrane region of this anion-selective channel. The studies here show that with the ability to sample multiple binding sites and coupled protein protonation states, MCCE provides a powerful tool to analyze and predict ion binding. The calculations overestimate the affinity of surface chloride in HSA and Omp32 relative to the buried ion in amylase. Differences between ion-solvent interactions for buried and surface ions will be discussed.     

The Structure and Diversity of α1-Acid Glycoprotein/Orosomucoid Gene in Africans

Human orosomucoid (ORM), or α₁-acid glycoprotein, is known to be controlled by duplicated and triplicated genes on chromosome 9, encoding ORM1 and ORM2 proteins. In this study, the structure and diversity of the ORM gene were investigated in 16 Sub-Saharan Africans, who originated from widely dispersed locations in Africa. The duplicated ORM1-ORM2 gene was observed in all 16 samples. ORM1*S1(2), characterized by an ORM2 gene-specific sequence in intron 5, was common in Africans. Three Africans showed the duplication of the ORM1 gene. The organization of the triplicated ORM1A-ORM1B-ORM2 gene was established in two Africans. The recombination breakpoints resulting in the ORM1 duplication lay within a small genomic interval around exon 1 of the ORM1B gene. The duplication of the ORM2 gene reported previously was not detected in this population sample. Several single-nucleotide polymorphisms were observed in the ORM2 gene. The rearrangement of the ORM gene is likely to occur often in Africans.     

The Binding of Benzopyranes to Human Serum Albumin. A Structure–Affinity Study

The binding of several benzopyranes to serum albumin was studied by equilibrium dialysis at pH7.4 in a 67mM sodium phosphate buffer at 37°C. The equilibrium data were analyzed using a computer program for curve fitting. The binding isotherm for warfarin, 4-hydroxycoumarin, 4-chromanol, coumarin, 3-acetylcoumarin, and benzoic acid can be described by two stoichiometric dissociation constants. Elimination of the 4-hydroxyl group in the coumarin chemical structures decreases the binding affinity of the compounds on the primary binding site of serum albumin, with 4-chromanol the smallest ligand which binds to seroalbumin with high affinity. Thus, the affinity of 4-benzopyranol and the 4-hydroxybenzopyranones greater than that of benzopyranones. On the other hand, elimination of the 2-oxo group in the benzopyranone chemical structures decreases affinity for the secondary binding site.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

Kinetic Considerations on the Development of Binding Assays in Single-Addition Mode: Application to the Search for α2δ1 Modulators

The development of assays in single-addition mode is of great interest for screening purposes given the multiple advantages of minimizing the number of intervention steps. Binding assays seem to be more prone to this attractive format because no functional biological activity is taking place but instead a biophysical process, whose dynamics seem easier to control without introducing significant alterations, is happening. Therefore, single-addition assays based on the displacement of prebound labeled ligands can be conceived, but careful kinetic considerations must still be taken to maximize the sensitivity of the assay and to avoid jeopardizing the identification of compounds with slow-binding kinetics. This article shows the development of a single-addition, displacement-based binding assay intended to identify modulators that act by binding to the gabapentin site of the ion channel regulatory protein α2δ1. After studying the kinetics of gabapentin binding and the influence they might have on the assay sensitivity, the best conditions were identified, and the sensitivity was compared with that of the more classical two-additions competition-based assay. Although the present study focuses on α2δ1 and its interaction with gabapentin, the rationale and the methodology followed are of broad purpose and can be applied to virtually every binding assay.     

Mapping the Interactions between the Alzheimer’s Aβ-Peptide and Human Serum Albumin beyond Domain Resolution

Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer’s disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494–515) region and aligns with the central hydrophobic core of Aβ. The HSA (495–515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association.Abbreviations: AD, Alzheimer’s Disease, BBB, Blood Brain Barrier, CNS, Central Nervous System, CSF, Cerebrospinal Fluid, FA, Fatty Acid, HSA, Human Serum Albumin, ICP, Inductively Coupled Plasma, MA, Myristic Acid, SL, Spin-Lock, RC, Random Coil, STD, Saturation Transfer Difference, STR, Saturation Transfer Reference, WG, Watergate water-suppression NMR technique     

Preferential and Specific Binding of Human αB-Crystallin to a Cataract-Related Variant of γS-Crystallin

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The 3��-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3′-extrahelical nucleotide (3′-flap) binding pocket. When presented with 5′-flap substrates having a 3′-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3′-flap on substrates reduced K_m and increased multiple- and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3′-flap, and the absence of a 3′-flap from a 5′-flap substrate was more detrimental to hFEN1 activity than removal of the 5′-flap or introduction of a hairpin into the 5′-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3′-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5′-phosphorylated product. Together the results indicate that the presence of a 3′-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA)⁶ two-way junction from which a 5′-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5′-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5′-flap structures are imprecisely cleaved by DNA2 in a replication protein A -dependent manner (7). Subsequent to the DNA2 cleavage, Rad27 (yeast homologue of FEN1) cleaves precisely to generate an intermediate suitable for ligation (2). The recent discovery that human DNA2 is predominantly located in mitochondria in various human cell lines (8, 9) suggests that hFEN1 is the paramount 5′-flap endonuclease in the nuclei of human cells. This observation potentially provides a plausible rationale for why deletion of RAD27 (yeast FEN1 homologue) is tolerated in Saccharomyces cerevisiae (10), whereas deletion of FEN1 in mammals is embryonically lethal (11). Recent models wherein mice carrying a mutation (E160D) in the FEN1 gene, which was shown in vitro to alter enzymatic properties (12), have demonstrated that FEN1 functional deficiency in mice (S129 and Black 6) increases the incidence of cancer, albeit different types presumably due to genetic background (13, 14). Thus, the function of mammalian FEN1 in vivo is vital to the prevention of genomic instability. In addition to its importance in the nucleus, hFEN1 has recently been detected in mitochondrial extracts (15, 16) and implicated in mitochondrial long patch base excision repair (15). Considering the pivotal roles of hFEN1 in DNA replication and repair, it is of interest to understand how hFEN1 and homologues achieve substrate and scissile phosphate selectivity in the absence of sequence information.Since its initial discovery as a nuclease that completes reconstituted Okazaki fragment maturation (17) and subsequent rediscovery as a 5′-flap-specific nuclease (DNaseIV) from bacteria (18), mouse (19), and HeLa cells (20), FEN1 proteins ranging from phage to human have been studied biochemically, computationally, and structurally (5, 6, 21). Biochemical characterizations of FEN1 proteins from various organisms have shown that this family of nucleases can perform phosphodiesterase activity on a wide variety of substrates; however, the efficiency of catalysis on various substrates differs among the species. For instance, phage FEN1s prefer pseudo-Y substrates (22, 23), whereas the archaeal and eukaryotic FEN1s prefer 5′-flap substrates (21, 24, 25), which have two dsDNA domains, one upstream and downstream of the site of cleavage, and a 5′-ssDNA protrusion (Fig. 1A). Primary sequence analysis indicates that FEN1 proteins share characteristic N-terminal (N) and Intermediate (I) “domains,” which harbor the highly conserved carboxylate residues that bind the requisite divalent metal ions (26–28). Structural studies of FEN1 nucleases from phage to humans (22, 29–36), have shown that the N and I domains comprise a single nuclease core domain consisting of a mixed, six- or seven-stranded β-sheet packed against an α-helical structure on both sides. The α-helices on either side of the β-sheet are “bridged” by a helical arch that spans the active site groove (supplemental Fig. S1). On one side of the β-sheet, the α-helical bundle (αb1) creates the floor of the active site and a DNA binding motif (helix-3-turn-helix) (32). Similarly, the opposite α-helical bundle (αb2) has also been observed to interact with DNA (35). Based on site-directed mutagenesis studies with T5 phage FEN1 (T5FEN1) (37) and hFEN1 (38, 39), and crystallographic studies of T4 phage FEN1 (T4FEN1) (22) and Archaeoglobus fulgidus FEN1 (aFEN1) (35) in complex with DNA, a general model for how FEN1 proteins recognize flap DNA has emerged. The helix-3-turn-helix motif is involved in downstream dsDNA binding, whereas the upstream dsDNA domain is bound by αb2. The helical arch is likely involved in 5′-flap binding (22).Open in a separate windowFIGURE 1.Secondary structure schematics of hFEN1 substrates. A, illustration of a general flap substrate created using a bimolecular approach whereby a template strand (T-strand), which partially folds into a hairpin, anneals with the duplex strand (d-strand). The T-strand hairpin creates the upstream dsDNA domain, whereas the d-strand base pairs with the T-strand to create the downstream dsDNA domain. The flap or any other structure is created by addition of nucleotides to the 5′-end of the d-strand. The interface between the upstream and downstream dsDNA domains may be viewed as a derivative of a two-way junction (74). Annealing of either the F(5), E, or G(15) d-strands with the T3F T-strand results in the formation of a (B) double flap substrate (Flap of 5-nt d-strand paired with a Template with a 3′-Flap, F(5)·T3F), C, exonuclease substrate with a 3′-extrahelical nucleotide (EXO d-strand paired with a Template with a 3′-Flap, E·T3F), and a D, fork-GEN substrate with a 3′-extrahelical nucleotide and a 15-nt ssDNA gap capped by a 23-nt hairpin structure (fork-Gap of 15-nt d-strand paired with a Template with a 3′-Flap, G(15)·T3F). E, annealing the F(5) d-strand with the T oligonucleotide creates a single flap (Flap of 5-nt d-strand paired with a Template, F(5)·T).Unlike phage FEN1s, studies of FEN1s from eubacterial (40), archaeal (21), and eukaryotic origins (41) have shown that the addition of a 3′-extrahelical nucleotide (3′-flap) to the upstream duplex of a 5′-flap substrate results in a rate enhancement and an increase in cleavage site specificity. Moreover, substrates possessing a 3′-flap, which mimic physiological “equilibrating flaps,” were cleaved exactly one nucleotide into the downstream duplex, thereby resulting in 5′-phosphorylated dsDNA product that was a suitable substrate for DNA ligase I (21, 41). As postulated by Kaiser et al. (21), the structure of an archaeal FEN1 in complex with dsDNA with a 3′-overhang showed that the protein contains a cleft adjacent to the upstream dsDNA binding site that binds the 3′-flap by means of van der Waals and hydrogen bonding interactions with the sugar moiety (35). Once the residues associated with 3′-flap binding were identified, sequence alignment analyses showed that the amino acid residues in the 3′-flap binding pocket are highly conserved from archaea to human. Furthermore, mutation of the conserved amino acid residues in the 3′-flap binding pocket of hFEN1 resulted in reduced affinity for and cleavage specificity on double flap substrates (42). Although the effects of the addition of a 3′-flap to substrates on hFEN1 catalysis are known qualitatively, a detailed understanding of the relationship between changes in catalytic parameters and rate enhancement by the presence of a 3′-flap is unknown. Here, we describe a detailed kinetic analysis of hFEN1 using four well characterized DNA substrates and show that the presence of a 3′-flap on a substrate not only contributes to substrate binding (42), but also increases multiple and single turnover rates of reaction in the presence of near physiological monovalent salt concentrations. We also demonstrate that, like T5FEN1, hFEN1 is rate-limited by product release, and thus multiple turnover rates at saturating concentrations of substrate are predominantly a reflection of product release and not catalysis as was previously concluded (39). Furthermore, this study provides insight into the mechanism of hFEN1 substrate recognition.     

Mapping the Interactions between the Alzheimer’s Aβ-Peptide and Human Serum Albumin beyond Domain Resolution

Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer’s disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494–515) region and aligns with the central hydrophobic core of Aβ. The HSA (495–515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association.     

Evidence for the Interaction between (−)-Epigallocatechin Gallate and Human Plasma Proteins Fibronectin,Fibrinogen, and Histidine-rich Glycoprotein

Human plasma proteins were subjected to affinity chromatography with (–)-epigallocatechin gallate (EGCg)-agarose, and the bound proteins were examined by sodium dodecylsulfate–polyacrylamide gel electrophoresis. A molecular weight evaluation of the protein bands suggested the presence of three proteins, fibronectin, fibrinogen, and a 75-kDa protein. When human serum was used, the 75-kDa protein dominated the bound fraction. The determination of the partial amino acid sequence of a peptide derived by endopeptidase digestion of this fraction suggested the 75-kDa protein to be histidine-rich glycoprotein (HRG). The presence of these proteins in the bound fraction was confirmed by the immunoblotting method. Affinity chromatography of the individual proteins indicated that fibrinogen and HRG had direct affinity for EGCg. Dot binding assays demonstrated the interaction of EGCg with these proteins. The method also showed that only gallate-containing catechins were bound by these proteins. These data suggest that when EGCg is absorbed in the body through the digestive system, it may interact with these proteins in blood plasma.     

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

Background

Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.

Methodology/Principal Finding

We demonstrate that stable cell lines inducibly expressing S1P-adapted α₁-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α₁-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α₁-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α₁-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.

Conclusions/Significance

Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.     

A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex

A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the α and σ2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on α-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the α-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on α-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex.CD4, a type I transmembrane glycoprotein that serves as a coreceptor for major histocompatibility complex class II (MHC-II) molecules, is expressed on the surfaces of helper T lymphocytes and cells of the monocyte/macrophage lineage (8). Primate immunodeficiency viruses gain access to these cells by virtue of the interaction of the viral envelope glycoprotein (Env) with a combination of CD4 and a chemokine receptor (63). This interaction causes a conformational change within the Env protein that promotes the fusion of the viral envelope with the plasma membrane. Upon the delivery of the viral genetic material into the cytoplasm of the host cells, one of the first virally encoded proteins to be expressed is Nef, an accessory factor that modulates specific signal transduction and protein-trafficking pathways in a manner that optimizes the intracellular environment for viral replication (reviewed in references 21, 39, and 65). Perhaps the best characterized function of Nef is the downregulation of CD4 from the surfaces of the host cells (6, 22, 29, 45). CD4 downregulation prevents superinfection (6, 41) and enhances virion release (19, 38, 48, 66, 76), thereby contributing to the establishment of a robust infective state (24, 72).The mechanism used by the Nef protein of human immunodeficiency virus type 1 (HIV-1) to downregulate CD4 has been the subject of extensive study, but only recently have the molecular details of this process begun to be unraveled. It is generally acknowledged that HIV-1 Nef accelerates the internalization of CD4 from the plasma membrane by linking the cytosolic tail of the receptor to the clathrin-associated endocytic machinery (1, 12, 20, 34, 40, 64). In support of this model, a hydrophobic pocket comprising W57 and L58 on the folded core domain of Nef binds with millimolar affinity to the cytosolic tail of CD4 (28) (all residues and numbers correspond to the NL4-3 variant of HIV-1 Nef used in this study). In addition, a dileucine motif (ENTSLL, residues 160 to 165) (10, 16, 26) and a diacidic motif (D174 and D175) (2) on the C-terminal flexible loop of Nef mediate an interaction of micromolar affinity with the clathrin-associated, heterotetrameric (α-β2-μ2-σ2) adaptor protein 2 (AP-2) complex (12, 20, 40, 49). These interactions draw CD4 into clathrin-coated pits that eventually bud inwards as clathrin-coated vesicles (11, 27). Internalized CD4 is subsequently delivered to endosomes and then to lysosomes for degradation (3, 23, 59, 64).Despite progress in the understanding of the mechanism of Nef-induced CD4 downregulation, several important aspects remain to be elucidated. Previous studies have shown that the Nef dileucine and diacidic motifs interact with a combination of the α and σ2 subunits of AP-2 (referred to as the α-σ2 hemicomplex) (12, 20, 40, 49), but the precise location of the Nef binding sites is unknown. It also remains to be determined whether Nef can actually bind CD4 and AP-2 at the same time. Indeed, the formation of a tripartite CD4-Nef-AP-2 complex in which Nef links the cytosolic tail of CD4 to AP-2 has long been hypothesized but has never been demonstrated experimentally. Given the relatively weak affinity of Nef for the CD4 tail (28) and AP-2 (12, 40), it is unclear how such a complex could assemble and function in CD4 downregulation.In this study, we have addressed these issues by using a combination of yeast hybrid, in vitro binding, and in vivo CD4 downregulation assays. We report the identification of a candidate binding site for the Nef diacidic motif on the AP-2 complex. This site, a basic patch comprising K297 and R340 on α-adaptin, is specifically required for Nef binding and Nef-induced CD4 downregulation. We also show that the Nef diacidic motif and the α-adaptin basic patch are required for the cooperative assembly of a tripartite complex composed of the CD4 cytosolic tail, Nef, and the α-σ2 hemicomplex. The cooperative manner in which this complex is formed explains how Nef is able to efficiently downregulate CD4 from the plasma membrane despite weak binary interactions between the components of this complex.     

Development of a High-Throughput Assay for Identifying Inhibitors of TBK1 and IKKε

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 μM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.