Conserved residues in the HIV-1 Nef hydrophobic pocket are essential for recruitment and activation of the Hck tyrosine kinase

The Nef protein of the primate lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is essential for high-titer viral replication and acquired immune deficiency syndrome (AIDS) progression. Nef binds to the macrophage-specific Src family member Hck through its SH3 domain, resulting in constitutive kinase activation capable of transforming rodent fibroblasts. Nef-Hck interaction may be essential for M-tropic HIV replication and AIDS pathogenesis, identifying this virus-host protein complex as a rational target for anti-HIV drug discovery. Here, we investigated whether interaction with Hck is a common feature of Nef alleles from different strains of HIV-1. We compared the ability of four different laboratory HIV-1 Nef alleles (SF2, LAI, ELI, and Consensus) to induce Hck activation and transformation in our Rat-2 fibroblast model. While SF2, LAI, and Consensus Nef all bound and activated Hck, ELI Nef failed to bind to the Hck SH3 domain in vitro and did not cooperate with Hck in fibroblast transformation. Molecular modeling identified three residues in the core region of SF2 Nef (Ala83, His116, and Tyr120) which are substituted in ELI with Glu, Asn, and Ile, respectively. Two of these residues (Ala83 and Tyr120) form part of the hydrophobic pocket that contacts Ile 96 in the RT loop of the Hck SH3 domain in the Nef-SH3 crystal structure. Substitution of SF2 Nef Tyr120 with Ile completely abolished Hck recruitment and activation. In a complementary experiment, substitution of ELI Ile120 with Tyr partly restored ELI Nef-induced Hck activation and transformation in Rat-2 cells. Hck activation increased further by substitution of ELI Glu83 with Ala and Asn116 with His, suggestive of a supportive role for these residues in Hck binding. This study provides the first biological evidence that the HIV-1 Nef hydrophobic pocket is critical to Hck recruitment and activation in vivo. Targeting the Nef hydrophobic pocket with a small molecule may be sufficient to disrupt Nef signaling through Hck in HIV-infected macrophages, slowing disease progression.     

Conformation of the dileucine-based sorting motif in HIV-1 Nef revealed by intermolecular domain assembly

The human immunodeficiency virus 1 (HIV-1) Nef protein is a pathogenicity factor required for effective progression to AIDS, which modulates host cell signaling pathways and T-cell receptor internalization. We have determined the crystal structure of Nef, allele SF2, in complex with an engineered SH3 domain of human Hck showing unnaturally tight binding and inhibitory potential toward Nef. This complex provides the most complete Nef structure described today, and explains the structural basis of the high affinity of this interaction. Intriguingly, the 33-residue C-terminal flexible loop is resolved in the structure by its interactions with a highly conserved hydrophobic groove on the core domain of an adjacent Nef molecule. The loop mediates the interaction of Nef with the cellular adaptor protein machinery for the stimulated internalization of surface receptors. The endocytic dileucine-based sorting motif is exposed at the tip of the acidic loop, giving the myristoylated Nef protein a distinctly dipolar character. The intermolecular domain assembly of Nef provides insights into a possible regulation mechanism for cargo trafficking.     

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.     

HIV-1 Nef control of cell signalling molecules: Multiple strategies to promote virus replication

HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. This small protein of approximately 27 kDa is required for maximal virus replication and disease progression. The mechanisms by which it is able to act as a positive factor during virus replication is an area of intense research and although some controversy surrounds Nef much has been gauged as to how it functions. Its ability to modulate the expression of key cellular receptors important for cell activation and control signal transduction elements and events by interacting with numerous cellular kinases and signalling molecules, including members of the Src family kinases, leading to an effect on host cell function is likely to explain at least in part its role during infection and represents a finely tuned mechanism where this protein assists HIV-1 to control its host.     

Dysregulation of autophagy by HIV-1 Nef in human astrocytes

Viruses often exploit autophagy, a common cellular process of degradation of damaged proteins, organelles, and pathogens, to avoid destruction. HIV-1 dysregulates this process in several cell types by means of Nef protein. Nef is a small HIV-1 protein which is expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HIV-Associated Neurocognitive Disorders (HAND). In order to explore its effect in the CNS with respect to autophagy, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus vector (Ad-Nef). We observed that Nef expression triggered the accumulation of autophagy markers, ATG8/LC3 and p62 (SQSMT1). Similar results were obtained with Bafilomycin A1, an autophagy inhibitor which blocks the fusion of autophagosome to lysosome. Furthermore co-expression of tandem LC3 vector (mRFP-EGFP-LC3) and Ad-Nef in these cells produced mainly yellow puncta (mRFP+, EGFP+) strongly suggesting that autophagosome fusion to lysosome is blocked in PHFA cells in the presence of Nef. Together these data indicate that HIV-1 Nef mimics Bafilomycin A1 and blocks the last step of autophagy thereby helping HIV-1 virus to avoid autophagic degradation in human astrocytes.     

Nef of HIV-1 interacts directly with calcium-bound calmodulin

It was recently found that the myristoyl group of CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is essential for the interaction between the protein and Ca(2+)-bound calmodulin (Ca(2+)/CaM). Based on the N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins, including the Nef protein from human immunodeficiency virus (HIV), we proposed a new hypothesis that the protein myristoylation plays important roles in protein-calmodulin interactions. To investigate the possibility of direct interaction between Nef and calmodulin, we performed structural studies of Ca(2+)/CaM in the presence of a myristoylated peptide corresponding to the N-terminal region of Nef. The dissociation constant between Ca(2+)/CaM and the myristoylated Nef peptide was determined to be 13.7 nM by fluorescence spectroscopy analyses. The NMR experiments indicated that the chemical shifts of some residues on and around the hydrophobic clefts of Ca(2+)/CaM changed markedly in the Ca(2+)/CaM-Nef peptide complex with the molar ratio of 1:2. Correspondingly, the radius of gyration determined by the small angle X-ray scattering measurements is 2-3 A smaller that of Ca(2+)/CaM alone. These results demonstrate clearly that Nef interacts directly with Ca(2+)/CaM.     

Large-scale purification and preliminary X-ray diffraction studies of human aspartylglucosaminidase

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous polypeptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 Å and are thus suitable for determination of the crystal structure of AGA.     

The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef''s dileucine motif LL_164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif–dependent manner. Treating HIV-1-infected CD4⁺ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4⁺ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4⁺ T cells.     

A Long Cytoplasmic Loop Governs the Sensitivity of the Anti-viral Host Protein SERINC5 to HIV-1 Nef

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Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane

The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.     

Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.     

Structure, dynamics, and Hck interaction of full-length HIV-1 Nef

Nef is an HIV accessory protein that plays an important role in the progression of disease after viral infection. It interferes with numerous signaling pathways, one of which involves serine/threonine kinases. Here, we report the results of an NMR structural investigation on full-length Nef and its interaction with the entire regulatory domain of Hck (residues 72-256; Hck32L). A helical conformation was found at the N-terminus for residues 14-22, preceding the folded core domain. In contrast to the previously studied truncated Nef (Nef Δ1-39), the full-length Nef did not show any interactions of Trp57/Leu58 with the hydrophobic patch formed by helices α1 and α2. Upon Hck32L binding, the N-terminal anchor domain as well as the well-known SH3-binding site of Nef exhibited significant chemical shift changes. Upon Nef binding, resonance changes in the Hck spectrum were confined mostly to the SH3 domain, with additional effects seen for the connector between SH3 and SH2, the N-terminal region of SH2 and the linker region that contains the regulatory polyproline motif. The binding data suggest that in full-length Nef more than the core domain partakes in the interaction. The solution conformation of Hck32L was modeled using RDC data and compared with the crystal structure of the equivalent region in the inactivated, full-length Hck, revealing a notable difference in the relative orientations of the SH3 and SH2 domains. The RDC-based model combined with (15)N backbone dynamics data suggest that Hck32L adopts an open conformation without binding of the polyproline motif in the linker to the SH3 domain.     

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.     

On the Solution Conformation and Dynamics of the HIV-1 Viral Infectivity Factor

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.     

Crystallization and preliminary X-ray diffraction studies of a bacterial flavohemoglobin protein

A flavohemoglobin protein (FHP) was isolated from Alcaligenes eutrophus and has been crystallized by vapor diffusion methods using PEG 3350 as precipitant. The crystals of the FAD- and heme-containing protein belong to the monoclinic space group P2₁ with unit cell parameters of 52.2 Å, 85.8 Å, 103.9 Å, and 81.8° corresponding to two molecules per asymmetric unit. The crystals diffract at least to a resolution of 2.0 Å and are suitable for an X-ray structure analysis. © 1995 Wiley-Liss, Inc.     

CIN85 associates with TNF receptor 1 via Src and modulates TNF-alpha-induced apoptosis

CIN85 is a multidomain protein that associates with receptors carrying tyrosine kinase domains. Here we report that it is also a component of the signaling complex associated with tumor necrosis factor receptor 1 (TNFR1), which lacks a tyrosine kinase domain. This was established by showing that CIN85 was co-precipitated with TNFR1, TRADD, cIAP-1 and TARF1/2, but not with FADD, RIP, caspase-8 or TRAF6. However, CIN85 did not bind directly to the cytoplasmic domain of TNFR1 (TNFR1-CYT) but to Src family kinases, Cbl and the p85alpha subunit of phosphatidylinositol 3-kinase (PI3-K p85alpha). Src bound directly to TNFR1-CYT, but Cbl and PI3-K p85alpha did not. A human cell line ectopically expressing CIN85 was 10 times more susceptible to TNF-alpha-induced apoptosis than control cells, which expressed identical levels of TNFR1 on their surface. However, the susceptibility of these two cell lines to CD95-induced apoptosis was the same. The three SH3 domains of CIN85 were essential for this increased susceptibility to apoptosis and its proline-rich regions were also required for maximal effect. TNF-alpha treatment recruited CIN85 to the TNFR1 signaling complex. Taken together, these results indicate that CIN85 associates with TNFR1 via Src and modulates TNF-alpha-induced apoptosis.     

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.     

Protein purification and preliminary crystallographic analysis of human Lyn tyrosine kinase

Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2 Å with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.     

Design and structural thermodynamic studies of the chimeric protein derived from spectrin SH3 domain

A number of the chimeric constructs with spectrin SH3 domain were designed for structural and thermodynamic studies of protein self-assembly and protein-ligand interactions. SH3 domains, components of many regulatory proteins, operate through weak interactions with proline-rich regions of polypeptide chains. The recombinant construct (WT-CIIA) studied in this work was constructed by linking the peptide ligand PPPVPPYSAG to the domain C-terminus via a long 12-residue linker to increase the affinity of this ligand for the spectrin domain, thereby ensuring a stable positioning of the polyproline helix to the conserved ligand-binding site in orientation II, which is regarded as untypical of the interaction between this domain and oligopeptides. A comparison of fluorescence spectra of the initial domain and the recombinant protein WT-CIIA suggests that the ligand sticks to the conservative binding site. However, analysis of the equilibrium urea-induced unfolding has demonstrated that this is an unstable contact, which leads to a two-stage unfolding of the chimeric protein. The protein WT-CIIA was crystallized; a set of X-ray diffraction data with a resolution of 1.75 Å was recorded from individual crystals. A preliminary analysis of these diffraction data has demonstrated that the crystals belong to space group P32 with the following unit cell parameters: a = b = 36.39, c = 112.17 Å, a = β = 90.0, and γ = 120.0.