The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

The p53 protein is a transcription factor that acts as the major tumor suppressor in mammals. The core DNAbinding domain is mutated in about 50% of all human tumors. The crystal structure of the core domain in complex with DNA illustrated how a single core domain specifically interacts with its DNA consensus site and how it is inactivated by mutation. However, no structural information for the tetrameric full-length p53-DNA complex is available. Here, we present novel experimental insight into the dimerization of two p53 core domains upon cooperative binding to consensus DNA in solution obtained by NMR. The NMR data show that the p53 core domain itself does not appear to undergo major conformational changes upon addition of DNA and elucidate the dimerization interface between two DNA-bound core domains, which includes the short H1 helix. A NMR-based model for the dimeric p53 core-DNA complex incorporates these data and allows the conclusion that the dimerization interface also forms the actual interface in the tetrameric p53-DNA complex. The significance of this interface is further corroborated by the finding that hot spot mutations map to the H1 helix, and by the binding of the putative p53 inhibitor 53BP2 to this region via one of its ankyrin repeats. Based on symmetry considerations it is proposed that tetrameric p53 might link non-contigous DNA consensus sites in a sandwich-like manner generating DNA loops as observed for transcriptionally active p53 complexes.The tumor suppressor gene p53 is the most frequent site of genetic alterations found in human tumors (1) and acts as the major tumor suppressor in mammals. In addition to non-transcriptional functions, p53 acts primarily as a transcriptional activator, that regulates the expression of several genes involved in cell cycle arrest, cellular senescence, anti-angiogenesis, and apoptosis (reviewed in Refs. 2-4). Recently, two homologues of p53, p63 and p73, were discovered, coding for a variety of different isoforms. These three p53 family members play distinct roles in differentiation, development, and tumor suppression (reviewed in Ref. 5). p53 possesses a modular architecture with an N-terminal transactivation domain (TAD), 1 a strongly conserved core DNA-binding domain (DBD), a tetramerization domain (TD), and a regulatory C terminus (6, 7). Tetrameric p53 binds specifically to a DNA consensus sequence consisting of two consecutive palindromic 10-bp halfsites, where each half-site is formed by two head-to-head quarter-sites (8 -12). The isolated TD forms a symmetric dimer of dimers (13-15), and contrasting models have been proposed that describe how the DBDs of each dimer are attached to DNA, namely with either consecutive or alternating arrangements (16). The p53 DBD comprises several hot spot regions for mutation that inactivate p53 in more than half of all human tumors (1). Therefore, wild-type and mutant p53 DBDs have been the focus of various studies (17-21). The crystal structure of the p53 DBD in complex with DNA (10) showed that almost all known...

The p53 protein is the major tumor suppressor in mammals. The discovery of the p53 homologs p63 and p73 defined a family of p53 members with distinct roles in tumor suppression, differentiation, and development. Here, we describe the biochemical characterization of the core DNA-binding domain of a human isoform of p63, p63-␦, and particularly novel features in comparison with p53. In contrast to p53, the free p63 core domain did not show specific binding to p53 DNA consensus sites. However, glutathione S-transferase-fused and thus dimerized p63 and p53 core domains had similar affinity and specificity for the p53 consensus sites p21, gadd45, cyclin G, and bax. Furthermore, the fold of p63 core was remarkably stable compared with p53 as judged by differential scanning calorimetry (T m ‫؍‬ 61°C versus 44°C for p53) and equilibrium unfolding ([urea] 50% ‫؍‬ 5.2 M versus 3.1 M for p53). A homology model of p63 core highlights differences at a segment near the H1 helix hypothetically involved in the formation of the dimerization interface in p53, which might reduce cooperativity of p63 core DNA binding compared with p53. The model also shows differences in the electrostatic and hydrophobic potentials of the domains relevant to folding stability.

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae mitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.The import of proteins into mitochondria is mediated by multisubunit translocases in the outer (TOM complex) and inner (TIM complex) membranes of the organelles (23,28,33). The TOM complex contains components which expose domains to the cytosol and act as preprotein receptors. The major import receptors are Tom20 and Tom22, which are essential for the specific recognition, unfolding, and translocation of the majority of preproteins (22). Both components interact with preproteins and cooperate in the formation of a presequence binding site termed the cis site (3,20,25,26,34). Another binding site for a more restricted set of preproteins, especially for members of the mitochondrial carrier family, is Tom70 (13,35,36), which acts in conjunction with Tom37 (12). From this binding site, preproteins are transferred to Tom20-Tom22 before entering the translocation pore (19).Other components of the TOM complex (Tom40, Tom5, Tom6, and Tom7) are deeply embedded in the outer membrane and are believed to form the translocation pore. Tom40 is an essential protein and was found in the vicinity of polypeptide chains in transit (31, 37, 39). The protein was suggested to be a central element of the preprotein-conducting pore of the mitochondrial outer membrane. The small members of the TOM complex are not essential by themselves, but combined deletion of their genes and those of other components of the translocase is lethal (1, 6, 15). Studies on the function of the small TOM complex proteins suggest that they play distinct roles. Tom6 and Tom7 were found to influence the stability of the TOM complex (1, 15). For Tom5 a function in facilitating preprotein transfer from the receptors into the translocati...

Protein kinase B (PKB)-selective inhibitors were designed, synthesized, and cocrystallized using the AGC kinase family protein kinase A (PKA, often called cAMP-dependent protein kinase); PKA has been used as a surrogate for other members of this family and indeed for protein kinases in general. The high homology between PKA and PKB includes very similar ATP binding sites and hence similar binding pockets for inhibitors, with only few amino acids that differ between the two kinases. A series of these sites were mutated in PKA in order to improve the surrogate model for a design of PKB-selective inhibitors. Namely, the PKA to PKB exchanges F187L and Q84E enable the design of the selective inhibitors described herein which mimic ATP but extend further into a site not occupied by ATP. In this pocket, selectivity over PKA can be achieved by the introduction of bulkier substituents. Analysis of the cocrystal structures and binding studies were performed to rationalize the selectivity and improve the design.

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound ef®ciently in a speci®c manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using¯uorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The af®nity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Background: Bispecific antibodies are currently emerging as a promising new class of cancer therapeutics. Results: The novel one-arm single chain Fab IgG bispecific antibody (XGFR) targeting IGF-1R and EGFR demonstrated potent signaling inhibition and enhanced ADCC induction. Conclusion: XGFR has shown in vitro and in vivo anti-tumor activity in pancreatic, lung, and colorectal mouse xenograft tumor models. Significance: Rational design can help to overcome low expression yields and impaired effector functions of bispecific antibodies.