TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by HIV-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm1,2. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice3,4 and several reports indicate that TRIM5 plays a role in signal transduction5–7, but the precise mechanism of restriction is unknown8. Here we demonstrate that TRIM5 promotes innate immune signaling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13/UEV1A, TRIM5 catalyzes the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (MAP3K7) kinase complex and stimulate AP-1 and NFκB signaling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13/UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NFκB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13/UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signaling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 A resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

AcrAB-TolC is the major efflux protein complex in Escherichia coli extruding a vast variety of antimicrobial agents from the cell. The inner membrane component AcrB is a homotrimer, and it has been postulated that the monomers cycle consecutively through three conformational stages designated loose (L), tight (T), and open (O) in a concerted fashion. Binding of drugs has been shown at a periplasmic deep binding pocket in the T conformation. The initial drug-binding step and transport toward this drug-binding site has been elusive thus far. Here we report high resolution structures (1.9–2.25 Å) of AcrB/designed ankyrin repeat protein (DARPin) complexes with bound minocycline or doxorubicin. In the AcrB/doxorubicin cocrystal structure, binding of three doxorubicin molecules is apparent, with one doxorubicin molecule bound in the deep binding pocket of the T monomer and two doxorubicin molecules in a stacked sandwich arrangement in an access pocket at the lateral periplasmic cleft of the L monomer. This access pocket is separated from the deep binding pocket apparent in the T monomer by a switch-loop. The localization and conformational flexibility of this loop seems to be important for large substrates, because a G616N AcrB variant deficient in macrolide transport exhibits an altered conformation within this loop region. Transport seems to be a stepwise process of initial drug uptake in the access pocket of the L monomer and subsequent accommodation of the drug in the deep binding pocket during the L to T transition to the internal deep binding pocket of the T monomer.

The multidrug exporter AcrB is the inner membrane component of the AcrAB-TolC drug efflux system in Escherichia coli and is responsible for the resistance of this organism to a wide range of drugs. Here we describe the crystal structure of the trimeric AcrB in complex with a designed ankyrin-repeat protein (DARPin) inhibitor at 2.5-Å resolution. The three subunits of AcrB are locked in different conformations revealing distinct channels in each subunit. There seems to be remote conformational coupling between the channel access, exit, and the putative proton-translocation site, explaining how the proton motive force is used for drug export. Thus our structure suggests a transport pathway not through the central pore but through the identified channels in the individual subunits, which greatly advances our understanding of the multidrug export mechanism.

Supplementary Table 1| Steady-state parameters for cleavage of 5-nitrobenzisoxazole by representative Kemp eliminases a Catalyst k cat (s-1) K M (mM) k cat /K M (M-1 s-1) (k cat /K M)/k AcO-b k cat /k uncat c Ref 34E4 d 0.66 0.12 5.5 x 10 3 9.5 x 10 7 5.7 x 10 5 11

Apoptosis is a vital mechanism in multicellular organisms to eliminate unwanted cells during development, tissue homeostasis, and immune system function (1). Initiation and regulation of apoptosis is highly controlled through specific proteinprotein interactions and by a family of proteolytic enzymes, the caspases (2, 3). One way to induce apoptosis is via death receptors, a subgroup of the tumor necrosis factor receptor superfamily (4). The death signal is transmitted through the binding of extracellular death ligands such as the Fas ligand (FasL) 1 to its receptor Fas resulting in conformational changes of preformed receptor clusters (5). Intracellularly this change leads to the recruitment of the adaptor protein FADD (6, 7) and of the initiator caspases, caspase-8 and -10 (8, 9). Fas and FADD interact via homophilic death domain interactions, whereas FADD and the pro-caspases interact through death effector domains (DED). Ligand, receptor, adaptor protein, and caspases form the death inducing signaling complex (DISC) (10). When recruited to the DISC, pro-caspase-8 or -10 is activated through a series of proteolytic cleavage steps. Activation of pro-caspases generally involves the cleavage within the proteolytic caspase domain, resulting in active caspase comprising a large (␣) and small (␤) subunit, as well as the removal of the N-terminal domain. Apoptosis by death receptors is regulated at different levels of the signaling pathway. The viral caspase inhibitors CrmA and p35 block caspase-8 once it is activated and released from the membrane-bound DISC (11). FLIP is a potent inhibitor of death receptor-mediated pro-apoptotic signals, blocking the signaling pathway more upstream, before caspase-8 activation and release (12)(13)(14)(15)(16)(17)(18)(19). Two forms, FLIP L (long form) and FLIP S (short form) have been characterized so far (20, 21), which correspond to FLIP splice variants at the mRNA level. FLIP S consists of two DEDs, whereas FLIP L has an additional C-terminal caspase domain and resembles caspase-8 in its overall structural organization. In the protease-like domain of FLIP L the catalytically active cysteine is replaced by a tyrosine rendering the molecule proteolytically inactive (20,21).Pro-caspase-8 and FLIP L are recruited to the DISC, where both molecules are partly processed and the cleaved intermediates remain bound to the DISC (12,22). In a recent paper, Krueger et al. (23) demonstrated that FLIP L but not FLIP S or a mutant lacking the small subunit of the protease domain contributes to the first cleavage step of caspase-8. It is assumed that, in both cases, caspase-8 activity is highly impaired, rendering cells resistant to death receptor-induced apoptosis (24).The precise physiological role of FLIP is still debated. Analysis of FLIP-deficient mice revealed not only its importance in the regulation of death receptor-induced apoptosis, but also in embryonic development (25). Cells deficient for FLIP are more susceptible to death receptor-mediated apoptosis (26), and this anti-apopto...

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ATP-binding cassette (ABC) transporters shuttle a wide variety of molecules across cell membranes by alternating between inward- and outward-facing conformations, harnessing the energy of ATP binding and hydrolysis at their nucleotide binding domains (NBDs). Here we present the 2.9-Å crystal structure of the heterodimeric ABC transporter TM287-TM288 (TM287/288) from Thermotoga maritima in its inward-facing state. In contrast to previous studies, we found that the NBDs only partially separate, remaining in contact through an interface involving conserved motifs that connect the two ATP hydrolysis sites. We observed AMP-PNP binding to the degenerate catalytic site, which deviates from the consensus sequence in the same positions as the eukaryotic homologs CFTR and TAP1-TAP2 (TAP1/2). The TM287/288 structure provides unprecedented insights into the mechanism of heterodimeric ABC exporters and will enable future studies on this large transporter superfamily.