Extended (red) and bent (gray) structures for GANG, YDD, and DALL3, respectively, were superimposed using their C-terminally adjacent domains. D, model of bending in the CheadCstalk structure. fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. Keywords: adhesin, bacterial adhesion, crystal structure, Gram-negative bacteria, membrane protein, protein domain, protein structure, structure-function, Acinetobacter, autotransporter == Introduction == In autotransporters, extracellular proteins in diverse Gram-negative bacteria, the transmembrane anchor domain (TM)2hosts the autotransport function, also called type V secretion, a process in which the passenger domain (PSD) is exported to the bacterial cell surface through a pore formed by the TM (1), with the assistance of periplasmic chaperones and the -barrel assembly machinery (2). Trimeric Rabbit Polyclonal to BTK (phospho-Tyr551) autotransporter adhesins (TAA) belong to a subfamily of the autotransporters, form homotrimeric structures with a common N-terminal head-stalk-membrane anchorC-terminal architecture (35). The head-stalk domain, a PSD of TAAs, is secreted by a C-terminal TM that is formed by a 12-stranded -barrel at the outer membrane (6). Although the TM is usually localized at the C terminus and is homologous in all TAAs and therefore defines this family, there are amino acid sequence alterations in the PSDs of SL 0101-1 many TAAs. The PSDs have a variety of lengths and mosaically arranged multiple domain structures that are distributed in many TAAs, and the daTAA program was developed for the annotation of TAA domains (7). TAAs adhere to host cells and/or extracellular matrix (ECM) proteins, such as collagen, fibronectin, and laminin, to mediate bacterial infection (3). Well known TAAs include YadA ofYersinia enterocolitica(8), BadA ofBartonella henselae(9), UspA1 and -A2 ofMoraxella catarrhalis(10), NhhA and NadA ofNeisseria meningitidis(1113), HadA, Hia, and Hsf ofHaemophilus influenzae(1416), and BpaA ofBurkholderia pseudomallei(17). Recently, many TAAs have been further identified, such as BtaE ofBrucellaspp. (18), Apa ofActinobacillus pleuropneumoniae(19), and EibD ofEscherichia coli(20). Ata ofAcinetobacter baumanniiis involved in nosocomial infection (21). SadA inSalmonellapromotes biofilm formation and host cell adherence (22). BimA inBurkholderiaspecies drives actin-based motility in host cells by mimicking host actin polymerases (23). The genusAcinetobacteris ubiquitously distributed in nature, such as in humans, animals, activated sludge, soil, water, and other environmental sources. For example , A. baumannii, Acinetobacter lwoffii, Acinetobacter parvus, Acinetobacter bereziniae, Acinetobacter guillouiae, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter pittii, andAcinetobacter nosocomialiswere isolated from clinical specimens (24). A. baumanniihas especially attracted our attention because it has caused nosocomial infection worldwide, and its multidrug-resistant strains have spread globally (25). The toluene-degrading bacteriumAcinetobactersp. Tol 5, an environmentally isolated nonpathogenic strain, exhibits an autoagglutinating nature and nonspecific high adhesiveness to both biotic, such as collagen, and abiotic material surfaces, from hydrophobic plastics to hydrophilic glass and stainless steel (26). This unique adhesive property is mediated by AtaA, the TAA of Tol 5 (27). Each polypeptide chain of AtaA comprises 3630 amino acid residues, and the homotrimer of the signal peptide-eliminated polypeptide forms a common configuration that can be broadly divided into five regions as follows: an N-terminal Ylhead domain (Nhead; 108315 aa); an N-terminal stalk (Nstalk; 3162904 aa); a C-terminal Ylhead domain (Chead; 29053169 aa); a C-terminal stalk (Cstalk; 31703561 aa); and a TM (35623630 aa) (Fig. 1A). Therefore , mature AtaA seems to be a short TAA comprising a complete set of domains, Chead-Cstalk-TM, to be fused with another set of PSDs, Nhead-Nstalk. In other words, AtaA seems to have two sets of PSDs that are tandemly fused, an N-terminal PSD (AtaA_NPSD, AtaA(59-2904)) and a C-terminal PSD (AtaA_CPSD, AtaA(29053561)). == FIGURE 1 . == Structure of the SL 0101-1 C-terminal part of the AtaA passenger domain. A, schematic representations of AtaA and of recombinant constructs from its C-terminal part (AtaA_CPSD): CheadCstalk (29053561 aa), Chead1 (29053168 aa), Chead2 (27773168 aa), CstalkFL (31703561 aa), CstalkN (31703332 aa), CstalkC1 (33343474 aa), and CstalkC2 (33343561 aa). All constructs were connected to GCN4 tags (white boxes). The Ylhead, FGG, GANG, Trp ring, DALL1, and GIN domains are labeled and numbered from the N terminus of full-length AtaA. Neck domains are not labeled. Pro-3061 of the CheadCstalk and Chead1 was mutated to Gly. Thenumbersabove the structures SL 0101-1 indicate amino acid residues. Signal peptide, YadA-like head, and transmembrane anchor, which are annotated by the daTAA program, are abbreviated asSP, Ylhead, andTM, respectively. The headCap was newly annotated from the solved crystal.