Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the 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.

In autotransporters, extracellular proteins in diverse Gramnegative bacteria, the transmembrane anchor domain (TM) 2 hosts 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 autotransporter adhesins (TAA) belong to a subfamily of the autotransporters, form homotrimeric structures with a common N-terminal headstalk-membrane anchor-C-terminal architecture (3)(4)(5). 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 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).
The genus Acinetobacter is 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, and Acinetobacter nosocomialis were isolated from clinical specimens (24). A. baumannii has especially attracted our attention because it has caused nosocomial infection worldwide, and its multidrugresistant strains have spread globally (25). The toluene-degrading bacterium Acinetobacter sp. 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; 108 -315 aa); an N-terminal stalk (Nstalk; 316 -2904 aa); a C-terminal Ylhead domain (Chead; 2905-3169 aa); a C-terminal stalk (Cstalk; 3170 -3561 aa); and a TM (3562-3630 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-(2905-3561)).
In this study, we crystallized new domains that are well conserved in TAAs, solved their crystal structures, and used them to determine the structure of AtaA_CPSD by modeling. In silico analysis revealed that AtaA_CPSD shares the common domain architecture of many Acinetobacter TAAs and that Ata of A. baumannii also has a similar domain architecture to Cstalk of AtaA in its C-terminal region. Therefore, functional analysis of this conserved region of AtaA was conducted to examine its contribution to the unique adhesive properties of AtaA and to expand our knowledge of the structural and functional features of Acinetobacter TAAs. In particular, we focused on the flexibility and toughness of the nanofibrous structure. Flexibility that allows TAA nanofibers to bend is considered important for exhibiting their adhesive function (10,20,22,28,29). However, if the nanofibers are too limp to extend to a target surface in an aqueous environment, TAAs cannot effectively interact with the surface. Resilience is required for fiber extension toward the target. Physical strength is also important for the AtaA nanofiber to resist shear stress and exhibit high adhesiveness without breaking the fiber. In addition, the trimeric structure of TAAs has been shown to be so stable as to be resistant to boiling in an SDS solution, which is essential for full-level adhesive activity of TAAs (30 -33). Here, we show the structure of AtaA_CPSD exhibiting toughness, which is mainly brought by interchain interactions and provides AtaA nanofibers with the resilience and physical strength that are important to exert high adhesiveness through the highly stable trimeric structure.
Crystallization and X-ray Crystallography-Proteins were first screened by a sitting drop vapor diffusion method at 20°C in 768 conditions using Classics Suite, Classics II Suite, PEGs Suite, PEGs II Suite, PACT Suite, JCSGϩ Suite, Protein Complex Suite, and Cryos Suite (Qiagen, Hilden, Germany). Protein and reservoir solutions (0.3 l each) were mixed using a Honeybee 963 crystallization robot (Genomic Solutions, Ann Arbor, MI) and periodically observed for 2 months under a Rock Imager (Formulatrix, Bedford, MA). Crystallization conditions were optimized by a hanging drop vapor diffusion method. The final crystallization conditions are listed in Table 1.
Crystals were soaked in buffers containing cryo-protectants, which were prepared by mixing the reservoir solutions for crystallization and various concentrations of cryo-protectants (Table 1). Crystals were loop-mounted and quenched in liquid nitrogen. Diffraction of the crystals was measured under cryoconditions at 100 K at the synchrotron beamline X10SA (PXII) of the SLS (Paul Scherrer Institute, Villingen, Switzerland) by using a PILATUS 6 M (DECTRIS, Baden, Switzerland), at the synchrotron beamline BL38B1 of the SPring-8 (Japan Synchrotron Radiation Research Institute) by using a CCD Quantum315r (ADSC, Poway, CA), or at the x-ray beamline at Nagoya University by using an imaging plate detector R-AXIS VII (RIGAKU, Akishima, Japan). Diffraction images were processed and scaled by using the XDS program suite or HKL2000 (36,37). The CCP4 program suite was used for model building and refinement (38). The phases of all structures were sequentially determined by molecular replacement using MOLREP (39). Chead1 and CstalkC1s were solved using UspA1 (PDB code 3NTN) and SadAK12 ((PDB code 2YO2) as templates of molecular replacement, respectively. CstalkFL was solved using SadAK1 (PDB code 2YNY) for FGG_5, CstalkC1i for YDD, and DALL3 domains, and BadA_head (PDB code 3D9X) for the GIN domain. CstalkN was solved using CstalkFL. After molecular replacement, novel structures of the headCap in the construct Chead1 and of the GANG domain in the construct CstalkFL could be traced and built automatically by ARP/wARP (40). All structures were manually modeled with Coot (41) and refined by REFMAC5 or PHENIX (42,43). Crystallographic parameters are summarized in Table 2.
Far-Western Blotting-ECMs (0.3 ng each) were separately applied onto all PVDF membrane sheets. As a positive control, one of the His-tagged recombinant proteins was applied onto each sheet. Once dried, 1 l of 30 M His-tagged recombinant proteins were applied onto the ECM spots and incubated for 30 min, followed by blocking with 5% skim milk. The bound proteins were immunodetected by a rabbit anti-His tag antibody and an HRP-conjugated anti-rabbit Ig antibody.
Cell Analysis-Electron microscopy, flow cytometry, adhesion assays, and autoagglutination assays were carried out as described previously (27) with slight modifications. For electron microscopy, cells were immunoreacted with the anti-Nhead antibody as first antibody and with colloidal gold (10 nm in diameter)-conjugated anti-rabbit IgG antibody as secondary antibody and observed under a transmission electron microscope (H-7600 HITACHI, Tokyo, Japan). For flow cytometry, cells were immunoreacted with the rabbit anti-Nhead antibody as first antibody and Alexa Fluor 488-conjugated anti-rabbit Ig antibody as secondary antibody. For confocal laser scanning microscopy (CLSM), cells were fixed with 4% paraformaldehyde for 15 min on a slide glass coated with gelatin, washed with phosphate-buffered saline (PBS), blocked with 2% BSA in PBS for 30 min, washed twice with NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, 0.05% Triton X-100, pH 7.6), and immunoreacted with the rabbit anti-Nstalk antibody for 10 min. After being washed twice with NET buffer, cells were immunoreacted with Alexa Fluor 488-conjugated anti-rabbit Ig antibody for 10 min. After further washing with NET buffer three times and deionized H 2 O once, cells were observed under

Crystallization of Domains of AtaA_CPSD
Using the daTAA program (7) and manual refinement (4,22), the domains of AtaA were reannotated, as shown in Fig. 1A. The Cstalk of AtaA was predicted to have FGG_4, GANG_10, GIN_15, YDD, DALL3, and FGG_5 from its N terminus (domains were numbered from the N terminus of AtaA) and eight coiled-coil segments (Fig. 2). The C-terminal Ylhead (Ylhead_2) was predicted to connect to an HANS motif and an unannotated region at its N-terminal end.
To determine the structure of AtaA_CPSD, we constructed seven redundant fragments that covered AtaA_CPSD as a series of recombinant proteins (Fig. 1A). Chead1 (2905-3168 aa), Chead2 (2777-3168 aa), CstalkN (3170 -3332 aa), and CstalkC1 (3334 -3474 aa) were designed to be connected to both N-and C-terminal trimeric GCN4 adaptors, GCN4pII, which were derived from a dimeric GCN4 leucine zipper (34). Such GCN4 fusions have been extensively used to stabilize coiled-coil domains of various proteins for structural studies (47). CheadCstalk (2905-3561 aa), CstalkC2 (3334 -3561 aa), and CstalkFL (3170 -3561 aa) carried the N-terminal GCN4 adaptor solely. We found that recombinant proteins of Chead1, Chead2, and CheadCstalk were partially degraded at Ylhead_2 (lanes WT in Fig. 1, B and C), although native AtaA on the Tol 5 cell surface was not degraded at the Ylhead_2 during cultivation. A proline residue sometimes causes such a partial degradation. Therefore, we introduced a point mutation of P3061G into the recombinant proteins. This mutation prevented the recombinant proteins from partial degradation (lanes P3061G in Fig. 1, B and C).
We successfully crystallized all of the recombinant constructs except for Chead2. Structures of Chead1 with a mutation of P3061G (PDB code 3WP8), CstalkFL (PDB code 3WPA), CstalkN (PDB code 3WPR), and CstalkC1 (PDB codes 3WPO, 3WPP, and 3WQA) were sequentially determined by molecular replacement. We did not solve the structures of CheadCstalk and CstalkC2. From the structures of Chead1, CstalkN, and CstalkFL, AtaA_CPSD (2905-3553 aa) was modeled (Fig. 1D). The last eight residues at the C-terminal coiled coil (3554 -3561 aa) are not included in this model, as they were not traceable in the electron density.
From this model, the length of AtaA_CPSD was estimated to be Ն450 Å long. Chead1 and CstalkFL followed crystallographic 3-fold symmetry and were therefore perfectly straight. The model suggested that HIM1 and FGG_4 are located close to each other. As seen in other TAAs (48), the coiled coils in Cstalk capture a Cl Ϫ ion and an unknown ion through Asn-3404 and Asp-3525, respectively. The right-handed coiled coil, which is often found in TAAs, was not observed in these structures of AtaA.
Unique Structure of Chead, Capped Ylhead-The crystal structure of the recombinant protein Chead1 revealed that its main domain, Ylhead, which is a trimer of left-handed ␤-helices composed of nine repeats of the 14 residues comprising a set of an inner face and an outer face, is capped by three ␤-hairpins and HANS, which were en bloc named headCap (Fig. 3). In addition, Ylhead_2 is inserted by HIM1 before its last inner ␤-strand. The headCap, Ylhead_2, and HIM1 integrally form Chead.
The structures of the Ylhead domains from YadA, BpaA, UspA1, EibD, and SadA have been solved previously (10,17,20,49), and of these, only the domain from BpaA is capped N-terminally, being preceded by tightly connected FGG and HANS motifs. This cap structure contains a central coiled-coil core, which carries the FGG motif as an insertion and ends in the HANS motif. As in all determined structures, the FGG motif moves the path of the chain by 120°counter-clockwise (ccw) around the trimer axis, as viewed from the N terminus. The Ylhead_2 of AtaA_CPSD is capped by a similar but architectur- ally more elaborate structure, the headCap, which has a number of unique features (Fig. 3). Like the BpaA cap, the headCap also consists of a helical core with coiled-coil character, which forms the central axis of the structure. However, it carries three ␤-hairpin insertions, only the last one of which is homologous to the last hairpin of FGG. Like FGG, the previous two hairpins also move the path of the chain by 120°ccw, but, being more elaborate, they offer considerably more options for interchain contacts. The HANS motif is also substantially extended at its C-terminal end relative to the BpaA cap, looping over by 120°c cw to form a cleft with the first (outer) strand of the following Ylhead domain. Sequence searches revealed that C-terminally extended HANS motifs always correlate with a preceding FGG motif that is truncated, as seen in AtaA. The HANS works as a connector between the C-terminal ␣-helix of the truncated FGG and the first strand of the Ylhead domain. The structural . 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. The numbers above the structures indicate amino acid residues. Signal peptide, YadA-like head, and transmembrane anchor, which are annotated by the daTAA program, are abbreviated as SP, Ylhead, and TM, respectively. The headCap was newly annotated from the solved crystal structure in this study and consists of ␤-hairpins, an N-terminally truncated FGG motif (⌬N-FGG), and a HANS motif. B, SDS-PAGE analyses of recombinant CheadCstalk proteins. CheadCstalk samples (WT and P3061G) purified by nickel-affinity chromatography were fractionated by SDS-PAGE and detected by Coomassie Blue staining (CBB). Only WT was sensitive to proteolysis, being degraded into 55-and 23-kDa fragments (WT fragment 55 and WT fragment 23, respectively), whereas the P3061G mutant was protease-resistant. C, Western blotting analyses of purified CheadCstalk samples using an anti-His tag antibody (IB). IB, immunoblot. The recombinant proteins carried the His tag at the C terminus, and only the 55-kDa fragment of the WT protein was detected. This confirms that cleavage had occurred in the region of Pro-3061 and that proline at this position represents a folding obstacle, presumably due to a necessary cis-trans isomerization step that overloads the endogenous prolyl isomerase during overexpression. D, three-dimensional structure of the AtaA_CPSD. The structures of Chead1, CstalkFL, CstalkN, and CstalkC1 were determined experimentally. CheadCstalk is a model constructed from these crystal structures. Of the three CstalkC1 structures (CstalkC1i (PDB code 3WPO); CstalkC1ii (PDB code 3WQA); and CstalkC1iii (PDB code 3WPP)), CstalkC1i is shown. In each structure, the three polypeptide chains forming a homotrimer are colored brown, green, and yellow, respectively; GCN4 tags are colored gray. Ions in CstalkC1i, CstalkFL, and CheadCstalk are shown as spheres: blue, chloride, and black, unidentified. Neck domains occur following the HIM1, GIN_15, YDD, and DALL3 domains. PDB code are indicated below the construct names.
elaboration of the AtaA headCap enables a large number of interchain contacts (Fig. 3, C-E), all of which are made to the preceding chain, 240°ccw around the trimer axis (Fig. 3B). Specifically, below, we review the interactions seen from the Ylhead domain toward the N terminus, listing pairwise interactions representatively for the 3-fold symmetric interactions along the fiber axis.
In the third ␤-hairpin, the Gln-2950 of chain A ( A ) is buried in a cleft formed by chain C ( C ) between the loop extending the HANS motif (2981 C -2985 C aa) and the first ␤-strand of Ylhead_2 (2989 C -2993 C aa) (Fig. 3E). Gln-2950 forms a hydrogen bond network consisting of its side chain carbonyl group interacting with the backbone nitrogen of Leu-2984 C and its side chain amino group interacting with the backbone carbonyl group of Asp-2989 C and with the side chain carbonyl group of Asn-3003 C , whose side chain amino group further anchors the side chain carbonyl groups of Asp-2983 C and Asn-3019 C . This network is additionally strengthened by Lys-2981 C , which interacts with the backbone carbonyl groups of Gly-2982 C and Gly-2985 C to provide rigidity to the HANS motif extension. Sequence searches show that the conserved glutamine of the third ␤-hairpin (Gln-2950 A ) and HANS motif extension have coevolved and only occur jointly (Fig. 2).
The third ␤-hairpin is capped by the second ␤-hairpin of the same chain (2929 A -2941 A aa) (Fig. 3D). Specifically, Asn-2930 A of the second ␤-hairpin interacts with the backbone nitrogen of Lys-2932 A , with the side chain of Asp-2954 A , and the backbone carbonyl group of Arg-2955 A in the third ␤-hairpin. In addition, Asn-2935 A of the second ␤-hairpin interacts with the backbone nitrogen of Asp-2954 A in the third ␤-hairpin. The third ␤-hairpin is anchored to the axis of the fiber at its N-terminal end by the bulky side chain of Phe-2942 A and by a captured water molecule between the backbones of Asn-2930 A in the second ␤-hairpin, Leu-2956 A in the third ␤-hairpin, and Gly-2917 of chain B ( B ) in the first ␤-hairpin.
The second ␤-hairpin itself is capped by the first ␤-hairpin (Fig. 3C). The two hairpins form a joint hydrophobic core consisting of Val-2914 C , Val-2922 C , Ile-2924 C , and Ile-2925 B ; Val-2928 B and Val-2929 B ; and a hydrogen bond network spanned by the water molecule captured by Asn-2930 B , Leu-2956 B , and Gly-2917 C .
Although the FGG-like third ␤-hairpin is found broadly among genera Acinetobacter, Neisseria, Moraxella, and Haemophilus, the second ␤-hairpin is not often found among TAAs. Proteins that are homologous to the second ␤-hairpin are only found in Psychrobacter aquaticus and Vitreoscilla stercoraria, in addition to genus Acinetobacter.
HIM1 is longer than HIM2 and HIM3, containing an ␣-helix instead of the ␤-hairpin of HIM2 or the loop of HIM3 (Fig. 4). HIM1 is anchored to the following neck and the following coiled coil through hydrophobic interactions with Leu-3117 and Val-3119, forming a layer of a hydrophobic core for the coiled coil.

Detailed Structures of the Domains in Cstalk
FGG Domain-The structure and amino acid sequence of the region containing the third ␤-hairpin of the AtaA_headCap (2942-2968 aa) are similar to those of the C-terminal half of the FGG domain of BpaA (16). The region (2912-2940 aa) of the AtaA_headCap corresponding to the N-terminal half of the FGG domain of BpaA is quite different and is no longer an FGG domain (Fig. 5, A and B). Therefore, we call it ⌬N-FGG. AtaA_ CPSD contains two FGG domains in Cstalk and a ⌬N-FGG in Chead. FGG_4 and FGG_5 are quite similar to the SadA FGG domain (22). LGG, the most frequent motif in this domain (4) FGG_4 and FGG_5 of AtaA_CPSD contact with their N-terminally neighboring domains HIM1 and the neck following DALL3, respectively, resulting in a continuous enlarged hydrophobic core (Fig. 5, D and E). Although the model of CheadCstalk shows that HIM1 and FGG_4 are situated closely to each other, no interaction was observed between them. However, the enlarged hydrophobic core of HIM1 continues due to the following FGG_4, which also forms a layer of a hydrophobic core for the coiled coil (Fig. 5D).
GANG Domain-The crystal structure of the GANG domain was first determined in this study. The GANG domain is topologically similar to the Trp ring domain, except for the absence of the first and second ␤-strands of the Trp ring (␤1Ј and ␤2Ј in Fig. 6), a short variant of the Trp ring, i.e. a truncated interleaved head (4). Therefore, the path of the chain of the GANG domain by topological crossover around its trimer axis is 40°c cw as viewed from the N terminus, compared with 120°ccw of that of Trp ring domain. Although the characteristic motif (Gly/Asn)-Ala-(Asp/Asn)-Gly of the GANG domain is often  Gly-Ala-Asn-Gly (GANG), it is NADG in AtaA_CPSD. This motif is positioned in the second loop between the second and third ␤-strands and is exposed outside of a trimer (Fig. 6B), suggesting that the loop interacts with substance surfaces. The GANG domain captures four water molecules around the connection between the N-terminal ␣-helix and the first ␤-strand of the GANG domain, suggesting that the GANG domain is more flexible than other TAA domains. Crystal structures showed high B-factors at the connection between FGG_4 and GANG_10 and at the NADG loop. The N-terminal ␣-helix does not interact with the GANG loop, probably also contributing to the flexibility of the loop and bending between the FGG and GANG domains. A phenylalanine residue in a Trp ring of HiaBD1 stabilizes trimerization of the Trp ring (50). The Trp ring of the BadA head also contains phenylalanine. The conserved Tyr in the GANG domain, which corresponds to the phenylalanine in the Trp ring, may stabilize trimerization of the GANG domain (Fig. 6E).
GIN Domain-GANG_10 is followed by a GIN domain in AtaA, as in other TAAs, in which GANG domains and Trp rings are generally connected at their C-terminal end to GIN or another interleaved head domain. The crystal structure of GIN_15 in AtaA_CPSD was confirmed to follow the general topology of GIN (4), which is a transversal head as well as Ylhead, and consists of a single, mainly antiparallel, ␤-sheet (Fig. 7B). The number of ␤-strands in the GIN domain varies. GIN_15 is composed of eight ␤-strands, which is two more than the GIN domain of BadA (Fig. 7A). The shortest GIN domain in AtaA is found as GIN_14, which is predicted to have three ␤-strands. In the ␤-meander of GIN_15, hydrophobic residues are regularly positioned inside for trimerization of the ␤-sheet to form a triangular prism (Fig. 7, C and D), which may stabilize AtaA through its large hydrophobic core.
DALL Domains-We determined the structures of two novel DALL variant domains that are conserved in TAAs, i.e. YDD and DALL3 in AtaA_CPSD. The DALL domain is composed of N-terminal ␣-helix and ␤-strands. In the structures of CstalkFL and CstalkC1, YDD is now seen to represent a divergent form of DALL2. The YDD domain does not have inserted loops containing an additional ␤-strand (␤Љ in Fig. 8A), resulting in a simple structure as the DALL2 domain (Fig. 8B). However, YDD has a tyrosine that is the conserved aromatic amino acid residue in the DALL1 domain (Fig. 8A, asterisk), whereas the tryptophan in the DALL2 domain of SadA at this position forms ainteraction with histidine across the chains (41st position in Fig. 8A) (22). Neither YDD nor DALL1 have the histidine residues, resulting in the absence of theinteraction in these domains in AtaA. In DALL3, only its C-terminal half is equivalent to DALL1 and DALL2/YDD; DALL3 contains two additional N-terminal strands. The N-terminal ␤-strand with GTVG forms a short ␤-strand on another ␤-strand with LVQQA (Fig. 8, A and C). Because of the additional N-terminal strands, the DALL3 domain transits a chain to axis different from other DALL domains (Fig. 8C). These additional ␤strands enclosed 13 water molecules in a trimer (Fig. 8D), which may make the N-terminal end of DALL3 more flexible than other DALL domains. Both YDD and DALL3 in AtaA_ CPSD are followed by a neck (Fig. 8, B and C), forming a globular and rigid domain that is topologically akin to a head domain, as usually seen in all DALL variants (4).

Bending of TAAs with DALL Domains
B-Factors at the connections between the N-terminal ␣-helix and first ␤-strand in the DALL variant domains were shown to be high. Furthermore, we solved crystal structures of CstalkC1 in various crystal forms, showing different bending angles at the YDD and DALL3 domains. CstalkC1ii was found to mostly bend; the trimer of CstalkC1ii bent 5°with the trimer axis of the DALL3-neck (Fig. 9, A and C). Because of this flexibility, the structure of the C terminus of CstalkC1iii was disordered, even in a crystal (PDB code 3WPP). As well as CstalkC1, the overall structure of CstalkN bent compared with that of CstalkFL (Fig.  1D). The trimer of CstalkN bent 1°at the root of the GANG domain (Fig. 9, B and C). Because of this flexibility, the N terminus of CstalkFL was disordered in a crystal (Fig. 1D). These structural flexibilities are considered to be caused by the absence of interactions between the C terminus of the ␣-helix and the first ␤-layer in the DALL3 and YDD domains and between the C terminus of the ␣-helix of FGG and the GANG loop in the GANG domain. On the basis of these structures, we constructed a bending model for CheadCstalk (Fig. 9D). This model shows that AtaA_CPSD bends a total of 6°from the C terminus.

Functional Analyses of AtaA_CPSD
To examine the contribution of AtaA_CPSD to the nonspecific high adhesiveness of AtaA, we first constructed three inframe deletion (IFD) mutants by deleting Chead and/or Cstalk from the full-length AtaA; they were IFD-⌬CheadCstalk (deletion from 2906 to 3475 aa), IFD-⌬Chead (deletion from 2902 to 3167 aa), and IFD-⌬Cstalk1 (deletion from 3170 to 3475 aa) contains an ␣-helix (␣). Hydrophobic residues are shaded gray. B, superimposition of HIM structures. AtaA_HIM1, BpaA_HIM2, UspA1_HIM2, and SadA_ HIM3 are colored in brown, green, yellow, and blue, respectively. The regions adjacent to HIMs, including a part of Ylhead, the neck, and the following ␣-helix, are colored gray. HIMs are superimposed using the following neck. (Fig. 10A). These constructs (IFD-AtaAs) were designed to connect the neck to the ␣-helix without breaking the secondary structure on the basis of the domain annotation on the amino acid sequence of AtaA (Fig. 11). After confirmation of the cell surface display of IFD-AtaAs by flow cytometry and CLSM (Fig.  10E), the states and appearances of their fibers extending from the cell surface were observed using immunoelectron microscopy (Fig. 10D). On IFD-⌬CheadCstalk-and IFD-⌬Chead-expressing cells, many extended fibers of the respective IFD-AtaAs were observed. The distal ends of these fibers, labeled with colloidal gold-conjugated anti-Nhead antibody, were observed ϳ200 nm apart from the cell surface. However, on IFD-⌬Cstalk1, the distal ends of many fibers were observed at the margin of the cells, despite the presence of a few extended fibers, suggesting that most of the fibers formed fell down or hardly extended or stood on the cells. The IFD-AtaA-expressing mutant cells were subjected to adherence assays for polystyrene (PS) and collagen surfaces and to autoagglutination assays. We found that IFD-⌬CheadCstalk-and IFD-⌬Cheadexpressing cells showed adhesiveness to PS and collagen surfaces and autoagglutination that were as high as those of WT cells (Fig. 10, G and H). In contrast, IFD-⌬Cstalk1-expressing cells showed greatly decreased adhesiveness and autoagglutination, probably due to the abnormally formed IFD-AtaA fibers mentioned above. Despite the decreased adhesiveness of this mutant, flow cytometry and CLSM showed that immunolabeling against Nhead was more intensive on IFD-⌬Cstalk1 than on WT AtaA (Fig. 10E). This could be explained by the increased reactivity of the antibody against this IFD construct in a partially unfolded state. After determination of the crystal structures of recombinant constructs from AtaA_CPSD, structure models of the new domain connection arising from the deletions in the IFDs were simulated (Fig. 10, B and C). This suggested that loops of HIM1 and FGG_5 slightly clash in IFD-⌬Cstalk1 because the intervening coiled coil was shortened too far, which would cause the formation of abnormal IFD-AtaA fibers. Therefore, we designed a new IFD, IFD-⌬Cstalk2 (deletion from 3177 to 3475 aa), in which the coiled coil was extended by one heptad. In this new construct, the collision of the loops was simulated to be avoided (Fig. 10C). The resultant cells displayed IFD-⌬Cstalk2 on the cell surface in the level similar to original AtaA on WT cells (Fig. 10E). The observed IFD-AtaA fibers normally extended, and their distal ends were ϳ200 nm distant from the cell surface (Fig. 10D). Cells of this IFD mutant exhibited adhesiveness to PS and collagen surfaces and autoagglutination that were as high as those of WT cells (Fig. 10, G and H). Thus, deletion of Chead and/or Cstalk had no effect on the adhesion or autoagglutination of Tol 5 cells. Then, we constructed IFD-CPSD, in which the region of AtaA(108 -2966) corresponding to most of the Nhead and Nstalk was deleted from the full-length AtaA and the N-terminal repeats of Ylhead_1 and Ylhead_2 were connected to each other, so as to generate truncated AtaA fibers mostly consisting of AtaA_CPSD. We confirmed that many short truncated AtaA fibers grew from IFD-CPSD cells by normal electron microscopy after negatively staining cells (Fig. 10F). However, IFD-CPSD cells lost adhesiveness to not only PS and collagen surfaces but also to fibronectin and laminin at a similar level as the ⌬ataA mutant (Fig. 10I). Finally, recombinant proteins whose crystal structure could be solved were subjected to a far-Western blotting assay against ECM proteins (Fig. 10J). Chead, CstalkN, and CstalkC2 did not bind to fibronectin, type I collagen, or laminin, whereas the head domain of YadA was used as a control bound to collagen and laminin. From all of the results of the adhesion assays, we concluded that the sites responsible for the nonspecific high adhesiveness of Tol 5 cells do not reside in AtaA_CPSD.

Domain Architecture of Acinetobacter TAAs
Proteins homologous to the C-terminal domains of AtaA (residues 2905-3630 or 3476 -3630) were identified by BLASTP. Of the 54 Acinetobacter species characterized so far, 23 species have a region homologous to AtaA_CPSD (Fig. 12). These TAAs can be separated into two types based on the presence or absence of Chead. In the former, the Chead does not reside at the N terminus of the protein, and there is an Nstalk at its N-terminal side as well as a Cstalk at its C-terminal side. In addition, these Cstalks have the same domain architecture as that of AtaA. Therefore, many Acinetobacter species have TAAs with a C-terminal region sharing common domain architecture with AtaA_CPSD. Among them, a hypothetical TAA of A. bereziniae has a CPSD that shows the highest homology with AtaA_CPSD; their amino acid sequences differ from each other at only three residues. The other types of TAAs have no Chead, but many of them have a C-terminal region with domain architecture similar to the Cstalk of AtaA. Ata of A. baumannii also belongs to this type, and only GANG_10 in the Cstalk of AtaA is substituted by the Trp ring in Ata at its corresponding site.  Fig. 1. The neck following the DALL3 and ␤-hairpins of FGG_5 project to the outside at 120 and 60°around the trimer axis, respectively. F, surface of models of AtaA_CheadCstalk, FGG (ϩ), and a truncation of ␤-hairpins in FGG_4 and FGG_5 of CheadCstalk, FGG (Ϫ). The surface is colored by distance from the trimer axis in a gradient from red to white and from white to green as shown by a scale bar.

Discussion
In this study, we determined, for the first time, the crystal structures of the HIM, GANG, YDD, and DALL3 domains, which are well conserved in TAAs. In addition, we newly iden-tified headCap and determined its unique structure. These structures add information to the "dictionary approach" to TAAs, based on which whole TAA fibers can be modeled on the basis of known fragments (22). Many Acinetobacter species have TAAs. In this study, it was revealed that AtaA_CPSD does not include a binding site responsible for high adhesiveness to various abiotic surfaces of full-length AtaA. This is reasonable because none of the other TAAs that have the same domain architecture as AtaA_CPSD exhibit similar adhesive properties to AtaA. The characteristic binding sites of AtaA are considered to reside in the unique AtaA_NPSD consisting of Nhead and Nstalk.

Structure of C-terminal Passenger of an
In the CPSD, however, we can see structurally important features that probably contribute to high adhesiveness, i.e. flexibility and toughness. For strong adhesion to targets that are distant from the cell surface, AtaA fibers must project their binding sites on Nhead and/or Nstalk sufficiently far from the cell surface. In fact, IFD-⌬Cstalk1 cells could not exhibit high adhesiveness because they formed fibers that hardly extended (Fig. 10). If the AtaA_CPSD at the base of the fiber is too limp to support the long fiber on the cell for extending beyond the cell surface, the interaction between the binding sites and target surfaces might be inefficient. Therefore, sufficient toughness to support the fiber and permit its extension is essential for TAAs to function as adhesins.
The toughness of TAAs largely depends on the interchain interactions of the three polypeptides forming the trimer. Hydrophobic core formation by residues on the inside of the trimer and topological chain exchange are mainly responsible for TAA toughness. GIN, FGG, and Ylhead domains significantly enlarge the hydrophobic core around the trimer axis. GIN and Ylhead domains are quite stable because of stacking of ␤-strands. FGG domains exert hydrophobic interactions between their ␤-hairpins and the coiled coil (Fig. 5C), contributing to toughness against horizontal shear stress. In addition, at the FGG domains, chain exchange occurs accurately 120 o ccw around the trimer axis (Fig. 5, C-E), contributing to toughness against vertical shear stress. There are many other chain interactions in AtaA_CPSD. In Chead and at the transitions of the FGG-GANG, GANG-GIN-neck, YDD-neck, and DALL3neck, polypeptide chains twist 120 or 240°ccw around the trimer central axis, resulting in complex chain exchange along the parallel line to the fiber axis. Of these, the headCap forms a markedly complex interaction network by frequent topological chain transitions in a short range of amino acid residues. A, sequence alignment of the 10 GANG domains of AtaA with two Trp ring domains, one from A. baumannii Ata, which substitutes in that protein for GANG_10, and the other from B. henselae BadA. Asterisks (bold) and carets (bold) indicate the conserved GANG and Tyr-polar-Val motifs, respectively. The characteristic tryptophan of Trp ring domains is highlighted in yellow. Residues forming the hydrophobic core are shaded gray. The ␤-strands of the domains are indicated by arrows, above the alignment for GANG and below for Trp ring. B, overall structure of GANG_10. Colors are as in Fig. 1. The GANG motif (NADG in this domain) is colored blue and shown in stick representation, as is the conserved tyrosine, Tyr-3256. Water molecules are shown as spheres; a water molecule at the trimer axis is colored red and the others gray. C, overall structure of the Trp ring domain, exemplified by the domain of B. henselae BadA (PDB code 3D9X). The conserved tryptophan and phenylalanine residues of the Trp ring domain are shown in stick representation. The phenylalanine is at the equivalent position to the conserved tyrosine of GANG domains. D, topological diagram of GANG and Trp ring domains, with loops as lines and ␤-strands as arrows. One chain of the trimer is colored, yellow for the part common between the GANG and Trp ring domains and black for the two additional ␤-strands of Trp ring domains (␤Ј1 and ␤Ј2). E, superimposition of single monomers from AtaA GANG_10 (brown) and BadA Trp ring (gray) obtained by structural alignment of the following GIN domains.  FEBRUARY 19, 2016 • VOLUME 291 • NUMBER 8  Fig. 1) and single YDD and DALL3 domains in the Cstalk. A histidine residue highly conserved in DALL2 domains is highlighted in yellow. A key aromatic residue of DALL1, DALL2, and YDD is indicated by an asterisk. DALL1 differs from DALL2/YDD by an extended ␤1-␤2 hairpin; in ␤2, the extension is separated from the rest of the ␤-strand by a ␤-bulge at a conserved tyrosine residue corresponding to Tyr-1325 in SadA, indicated by a caret and marked as ␤Љ in the figure. The N-terminal half of DALL3, shown in lowercase letters, is topologically different from DALL1 and DALL2/YDD, containing two additional ␤-strands (␤1Ј and ␤2Ј) and orienting ␤1 in the opposite direction. Residues forming the hydrophobic core are shaded gray. B, structures of DALL domain variants and the following necks. DALL1 and DALL2 are from SadA ((PDB code 2YO3 and 2YNZ, respectively); YDD and DALL3 are from CstalkFL. In each structure, the main chain of one subunit forming the DALL domain is shown in color and stick representation. Water molecules interacting with this subunit are indicated by spheres of the same color. In DALL3, water molecules interacting with the other subunits are colored black. Water molecules along the 3-fold axes of DALL1, DALL2, and DALL3 are colored red. YDD alone does not contain an axial water molecule, allowing the central water molecule of the following neck to be visible (colored in white). C, superimposition of DALL domain monomers, obtained by structural alignment of the following neck domains. The residues of the two additional ␤-strands in DALL3 are labeled. D, hydrogen bond network of water molecules in DALL3, showing the 13 water molecules of a trimer in the same colors as in B. Hydrogen bonds are indicated by dotted lines. ously with HIM1 so as not to cause stress concentration at this point. The coiled coil at this region is elongated by one heptad insertion to avoid the collision between the HIM1 loop and the FGG loop. Slight collision of the loops would not be allowed to form the normal extended fibers in the IFD-⌬Cstalk1, demonstrating the exquisite design of AtaA in nature.

Structure of C-terminal Passenger of an Acinetobacter TAA
The importance of flexibility for adhesion has also been pinpointed for different TAAs (10,20,22,28,29). Bending of TAA fibers allows them to face and angle their binding sites to target surfaces, especially to large receptors, matrix proteins, or abiotic surfaces with a large radius of curvature, for effective interaction. The angle of bending is estimated at about 90°by the summation of DALL1 (8 ϫ 10°from SadA K14), FGG-GANG (4 ϫ 1°from CstalkN), and a combination of YDD and DALL3 (1 ϫ 5°from CstalkCii) (Fig. 9E). In fact, we observed that AtaA bent flexibly on the cell surface (Fig. 9F).
Ions are often captured by hydrophilic residues, one of which is often asparagine at position d of the heptad coiled coil of TAAs and is therefore called the N@d motif (48). Cstalk has Asn-3404 at position d and Asp-3525 at position a to capture Cl Ϫ and an unknown ion, respectively. The latter was not included in the final model, but we suspected it to be Mg 2ϩ because of its coordination geometry. These ions may also give Cstalk flexibility by breaking the hydrophobic core.  Fig. 1. Sequences are labeled with the species and strain name of the source organism, and the sequence ID in the NCBI nr database. AcTAA, Acinetobacter TAA.
In DALL domains, flexibility and toughness are compatibly exhibited; the former comes from the connection between N-terminal ␣-helix and the first ␤-strand and the latter from topological chain exchange at the connection to the neck and a hydrophobic core. Note that DALL necks are often found in relatively long TAAs with over 1000 residues. TAAs may have evolved themselves to achieve flexibility and toughness, which appear opposite features, by scattering DALL-neck insertions along coiled-coil fibers, such as in AtaA, which has 10 repeats of this structure.
Although AtaA_CPSD is not essential for the nonspecific high adhesiveness of Tol 5 cells, its structure is exquisite for exhibiting both flexibility and toughness, which provide the resilience needed by full-length AtaA fibers to exert their adhesive properties across a wide range of conditions.