Mechanistic insights into the activation of the IKK kinase complex by the Kaposi's sarcoma herpes virus oncoprotein vFLIP.

Constitutive activation of the canonical NF-κB signaling pathway is a major factor in Kaposi's sarcoma-associated herpes virus pathogenesis where it is essential for the survival of primary effusion lymphoma. Central to this process is persistent upregulation of the inhibitor of κB kinase (IKK) complex by the virally encoded oncoprotein vFLIP. Although the physical interaction between vFLIP and the IKK kinase regulatory component essential for persistent activation, IKKγ, has been well characterized, it remains unclear how the kinase subunits are rendered active mechanistically. Using a combination of cell-based assays, biophysical techniques, and structural biology, we demonstrate here that vFLIP alone is sufficient to activate the IKK kinase complex. Furthermore, we identify weakly stabilized, high molecular weight vFLIP-IKKγ assemblies that are key to the activation process. Taken together, our results are the first to reveal that vFLIP-induced NF-κB activation pivots on the formation of structurally specific vFLIP-IKKγ multimers which have an important role in rendering the kinase subunits active through a process of autophosphorylation. This mechanism of NF-κB activation is in contrast to those utilized by endogenous cytokines and cellular FLIP homologues.

A structurally conserved motif in γ-herpesvirus uracil-DNA glycosylases elicits duplex nucleotide-flipping.

Efficient γ-herpesvirus lytic phase replication requires a virally encoded UNG-type uracil-DNA glycosylase as a structural element of the viral replisome. Uniquely, γ-herpesvirus UNGs carry a seven or eight residue insertion of variable sequence in the otherwise highly conserved minor-groove DNA binding loop. In Epstein-Barr Virus [HHV-4] UNG, this motif forms a disc-shaped loop structure of unclear significance. To ascertain the biological role of the loop insertion, we determined the crystal structure of Kaposi's sarcoma-associated herpesvirus [HHV-8] UNG (kUNG) in its product complex with a uracil-containing dsDNA, as well as two structures of kUNG in its apo state. We find the disc-like conformation is conserved, but only when the kUNG DNA-binding cleft is occupied. Surprisingly, kUNG uses this structure to flip the orphaned partner base of the substrate deoxyuridine out of the DNA duplex while retaining canonical UNG deoxyuridine-flipping and catalysis. The orphan base is stably posed in the DNA major groove which, due to DNA backbone manipulation by kUNG, is more open than in other UNG-dsDNA structures. Mutagenesis suggests a model in which the kUNG loop is pinned outside the DNA-binding cleft until DNA docking promotes rigid structuring of the loop and duplex nucleotide flipping, a novel observation for UNGs.

IKKγ-Mimetic Peptides Block the Resistance to Apoptosis Associated with Kaposi's Sarcoma-Associated Herpesvirus Infection.

Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-κB pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IκB kinase (IKK) kinase, IKKγ (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP-IKKγ interaction both in cells and in vitro can be achieved using IKKγ-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKKγ complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.

KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing.

Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated.

Molecular basis of ion permeability in a voltage-gated sodium channel.

Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) â‰« K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.

Distinct Activation Mechanisms of NF-κB Regulator Inhibitor of NF-κB Kinase (IKK) by Isoforms of the Cell Death Regulator Cellular FLICE-like Inhibitory Protein (cFLIP).

The viral FLICE-like inhibitory protein (FLIP) protein from Kaposi sarcoma-associated herpesvirus activates the NF-κB pathway by forming a stable complex with a central region (amino acids 150-272) of the inhibitor of NF-κB kinase (IKK) γ subunits, thereby activating IKK. Cellular FLIP (cFLIP) forms are also known to activate the NF-κB pathway via IKK activation. Here we demonstrate that cFLIPL, cFLIPS, and their proteolytic product p22-FLIP all require the C-terminal region of NEMO/IKKγ (amino acids 272-419) and its ubiquitin binding function for activation of the IKK kinase (or kinase complex), but none form a stable complex with IKKγ. Our results further reveal that cFLIPLrequires the linear ubiquitin chain assembly complex and the kinase TAK1 for activation of the IKK kinase. Similarly, cFLIPSand p22-FLIP also require TAK1 but do not require LUBAC. In contrast, these isoforms are both components of complexes that incorporate Fas-associated death domain and RIP1, which appear essential for kinase activation. This conservation of IKK activation among the cFLIP family using different mechanisms suggests that the mechanism plays a critical role in their function.

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit ß by EPR Spectroscopy.

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/ß subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKß is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.

Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes.

UvrB has a central role in the highly conserved UvrABC pathway functioning not only as a damage recognition element but also as an essential component of the lesion tracking machinery. While it has been recently confirmed that the tracking assembly comprises a UvrA2B2 heterotetramer, the configurations of the damage engagement and UvrB-DNA handover complexes remain obscure. Here, we present the first crystal structure of a UvrB dimer whose biological significance has been verified using both chemical cross-linking and electron paramagnetic resonance spectroscopy. We demonstrate that this dimeric species stably associates with UvrA and forms a UvrA2B2-DNA complex. Our studies also illustrate how signals are transduced between the ATP and DNA binding sites to generate the helicase activity pivotal to handover and formation of the UvrB2-DNA complex, providing key insights into the configurations of these important repair intermediates.

Kaposi's sarcoma-associated herpesvirus vFLIP and human T cell lymphotropic virus type 1 Tax oncogenic proteins activate IkappaB kinase subunit gamma by different mechanisms independent of the physiological cytokine-induced pathways.

Activation of IκB kinase subunit γ (IKKγ), a key regulator of the classical NF-κB pathway, by the vFLIP protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and the Tax protein of human T cell lymphotropic virus type 1 (HTLV1) is essential for virus-associated cancer. We show that vFLIP and Tax activate this pathway by different interactions with IKKγ and independently of the ubiquitin-mediated signaling pathways induced by cytokines. Our data provide new insights into the mechanisms by which IKKγ can be activated and show that NF-κB activation by oncogenic viruses can be targeted without affecting physiologically important pathways.

Crystal structure of a KSHV-SOX-DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff.

The early lytic phase of Kaposi's sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined. We present the first crystal structure of a KSHV SOX-DNA complex that has illuminated the catalytic mechanism underpinning both its endo and exonuclease activities. We further illustrate that KSHV SOX, similar to its Epstein-Barr virus homologue, has an intrinsic RNase activity in vitro that although an element of host shutoff, cannot solely account for the phenomenon.

Crystal structure of a vFlip-IKKgamma complex: insights into viral activation of the IKK signalosome.

Key to the pathogenicity of several viruses is activation of the canonical nuclear factor-kappaB (NF-kappaB) transcriptional pathway. Subversion of this tightly regulated mechanism is achieved through the production of host mimetic viral proteins that deregulate the transcription process. One such protein is ks-vFLIP (produced by the Kaposi's sarcoma herpes virus [KSHV]), which associates with IKKgamma, an essential component of the IKK complex or signalosome. This interaction renders the canonical NF-kappaB pathway constitutively active and has been linked to Kaposi's sarcoma and other malignancies. In order to elucidate the molecular basis underpinning ks-vFLIP-induced activation of the IKK signalosome, we have determined the crystal structure of a complex involving a fragment of IKKgamma bound to ks-vFLIP at 3.2 A. In addition to identifying and subsequently probing the ks-vFLIP-IKKgamma interface, we have also investigated the effects of a mutation implicated in the genetic disorder anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID).

Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida.

Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, alpha and beta; the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the alpha subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase alpha subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the alpha subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.