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.

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.

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...

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.

No abstract