We provide here the first evidence that CarO channels possess an imipenem (but not meropenem) binding site, and that their specificities depend on their primary structure. Any decrease in CarO expression would thus reduce the susceptibility of A. baumannii to this antibiotic.

A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.

␤-TrCP is the F-box protein component of an Skp1/ Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that ␤-TrCP targets the oncogenic protein ␤-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from ␤-catenin containing the phosphorylated motif DpSGXXpS (P-␤-Cat 17-48 ) and ␤-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the ␤-TrCP binding pocket. The amide and aromatic protons of the His 24 and Trp 25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter ␤-catenin fragment with the ␤-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated ␤-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.The ubiquitin-proteasome pathway of protein degradation is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction (1, 2). This work is based on the study of the oncogenic protein ␤-catenin (␤-Cat), 1 which plays an essential role in the Wingless/Wnt signaling pathway and is an important component of cadherin cell-adhesion complexes (Fig. 1A). The abundance of ␤-catenin in the cytoplasm is regulated by ubiquitindependent proteolysis (3), and Wnt signaling is regulated by the presence or absence of the intracellular protein ␤-catenin. When Wnt signal is absent, the signal transduction pathway is off because ␤-catenin is rapidly destroyed. A large multiprotein machine normally facilitates the addition of phosphate groups to ␤-catenin by glycogen synthase kinase-3␤ (GSK3␤). Phosphorylated ␤-catenin binds to a protein called ␤-TrCP and is then modified by the covalent addition of a small protein called ubiquitin. Proteins tagged with ubiquitin are degraded by the 26 S proteasome, the protein-recycling center of the cell. When cells are exposed to the Wnt signal, it binds to cell surface receptors. Receptor activation blocks ␤-catenin phosphorylation, and its subsequent ubiquitination by an unknown mechanism that requires the intervention of the Disheveled protein (Dsh). ␤-Catenin is thus diverted from the proteasome. It accumulates and enters the nucleus, where it finds a par...

Edited by Miguel De la RosaThis study was undertaken to characterize functions of the outer membrane protein OmpW, which potentially contributes to the development of colistinand imipenem-resistance in Acinetobacter baumannii. Reconstitution of OmpW in artificial lipid bilayers showed that it forms small channels (23 pS in 1 M KCl) and markedly interacts with iron and colistin, but not with imipenem. In vivo, 55 Fe uptake assays comparing the behaviours of DompW mutant and wild-type strains confirmed a role for OmpW in A. baumannii iron homeostasis. However, the loss of OmpW expression did not have an impact on A. baumannii susceptibilities to colistin or imipenem.

The replication cycle of human immunodeficiency virus, type 1 (HIV-1) 4 involves the insertion of a DNA copy of its RNA genome into a chromosome of the host cell. Following retrovirus entry, a large nucleoprotein complex called preintegration complex (PIC) is formed in the cytoplasm with components of the virion core and cellular factors. In addition to viral cDNA, PICs contain several viral proteins: matrix, nucleocapsid, reverse transcriptase, VPR protein, and integrase (IN), which carries out DNA-cutting and -joining reactions (1, 2). HIV-1 IN consists of three functional domains: the N-terminal domain (residues 1-49), the catalytic core domain (residues 50 -212), and the C-terminal domain (residues 213-288) (3, 4). The N-terminal domain contains an HHCC motif that binds one Zn 2ϩ atom and is involved in the multimerization of the protein (4). The C-terminal domain binds DNA non-specifically and plays a role in the formation of an active multimer of IN (3). The catalytic core domain contains the canonical 3-amino acid motif, D, D(35)E, that is essential for the catalytic activity of the protein (4). These residues coordinate a divalent metal ion (Mg 2ϩ ) and are highly conserved among all integrases and retrotransposases. Integration proceeds in three steps, 3Ј-processing, strand transfer, and gap repair. Purified IN exhibits both 3Ј-processing and strand transfer in vitro. Double-stranded blunt-ended viral DNA produced by reverse transcription is first cleaved immediately 3Ј of a conserved CA dinucleotide motif. This reaction generates CA-3Ј-hydroxyl DNA ends that are the active intermediates of the strand transfer reaction. Both viral DNA ends are then inserted into a host cell chromosome. Finally, gap filling of the unrepaired 5Ј-ends of the viral DNA is under the control of cellular enzymes. In addition to the integration reaction, non-homologous end-joining and homologous recombination cellular pathways are involved in the formation of 1 and 2-LTR circles that are detected in the nuclei of infected cells (5-7).Cellular factors, such as SNF5/Ini1 (8), LEDGF/p75 (9 -14), EED (15), Rad18 (16), and HSP60 (17), were characterized to interact directly with IN. Other cellular proteins, such as HMG1a and Barrier of Autointegration Factor (BAF) interact with viral cDNA and participate in the integration reaction (18,19). While in vivo HIV-1 integration is not sequence specific, transcriptionally inactive regions of the genome, such as centromeres and telomeres, are disfavored targets (20 -22). Integration of proviral HIV-1 DNA occurs preferentially into transcriptional units of active genes, whereas the oncoretrovirus * This work was supported in part by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), Sidaction, Association Pour la Recherche sur le Cancer, and by EC project "Hidden HIV Challenge" (FP6-2003-LIFESCI-HEALTH-3/012182) (to S. E.). The Benarous and Mouscadet laboratories are supported by EC project TRIoH (LSHB-CT-2003-503480). The costs of publication of this article were defrayed in ...

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