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