SignificanceNosocomial infections and infections of indwelling devices are major healthcare problems worldwide. These infections are strongly associated with the ability of pathogens to form biofilms on biotic and abiotic surfaces. Panantibiotic-resistant Acinetobacter baumannii is one of the most troublesome pathogens, capable of colonizing medical devices by means of Csu pili, an adhesive organelle that belongs to the widespread class of archaic chaperone–usher pili. Here, we report an atomic-resolution insight into the mechanism of bacterial attachment to abiotic surfaces. We show that archaic pili use a binding mechanism that enables bacterial adhesion to structurally variable substrates. The results suggest a simple and cheap solution to reduce infections of A. baumannii and related pathogens.

Crystal structures of native and a-D-galactose-bound Bacillus circulans sp. alkalophilus b-galactosidase (Bca-b-gal) were determined at 2.40 and 2.25 Å resolutions, respectively. Bca-b-gal is a member of family 42 of glycoside hydrolases, and forms a 460 kDa hexameric structure in crystal. The protein consists of three domains, of which the catalytic domain has an (a ⁄ b) 8 barrel structure with a cluster of sulfur-rich residues inside the b-barrel. The shape of the active site is clearly more open compared to the only homologous structure available in the Protein Data Bank. This is due to the number of large differences in the loops that connect the C-terminal ends of the b-strands to the N-terminal ends of the a-helices within the (a ⁄ b) 8 barrel. The complex structure shows that galactose binds to the active site as an a-anomer and induces clear conformational changes in the active site. The implications of a-D-galactose binding with respect to the catalytic mechanism are discussed. In addition, we suggest that b-galactosidases mainly utilize a reverse hydrolysis mechanism for synthesis of galacto-oligosaccharides. DatabaseThe coordinates for free and a-D-galactose-bound Bca-b-gal structures have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under accession codes 3TTS and 3TTY, respectively.

Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.

Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A-and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligandbinding domain at 2.3 Å resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.