TamA is an Escherichia coli Omp85 protein involved in autotransporter biogenesis. It comprises a 16-stranded transmembrane β-barrel and three POTRA domains. The 2.3-Å crystal structure reveals that the TamA barrel is closed at the extracellular face by a conserved lid loop. The C-terminal β-strand of the barrel forms an unusual inward kink, which weakens the lateral barrel wall and creates a gate for substrate access to the lipid bilayer.

regions of MAS are centrally connected by non-conserved linkers, which permit large-scale 10 relative motions in related systems 10 . To obtain a high-quality hybrid model, we divided MAS 11 into its condensing and modifying region, and excluded the flexibly tethered ACP (Fig. 1a). 12Three constructs of staggered C-terminal length were employed to define the length of 13 the condensing region (see Methods). All variants crystallized under the same condition; 14 structure determination mapped the last ordered residue to Glu887. The structure of the most 15 extended variant (1-892) was refined at 2.3 Å resolution (Extended Data Table 1a). MAS KS-AT 16 comprises an α/β-fold linker domain (LD) connecting AT to KS (Fig. 1b) Fig. 1a-c). Four interface segments of 6-19 amino acid (aa) length are disordered 8 (Fig. 1b), while equivalent regions are ordered in dimeric KS domains. connect to the modifying region, are proximal to the two-fold dimer axis above the KS active 15 site, as observed in previous condensing region structures 6,11,12 . 16The DHs connect the modifying region to the post-AT linkers of the condensing region. 17We solved crystal structures of a MAS DH construct (aa 884-1186), which overlaps in sequence 18 with the crystallized KS-AT, in two crystal forms with a total of six protomers arranged into 19 almost identical dimers (Extended Data Table 1a). The DH protomer is composed of two hot-dog 20 folds connected by a 20 aa hot-dog linker (Fig. 1c). A hydrophobic substrate binding tunnel 21 extends over both hot-dog folds with entrances near the C-terminus and at the distal end of hot-22 dog fold 2. Active site residues are contributed by both hot-dog folds and are located close to the Table 2a). In the DH dimer, the two protomers arrange 2 with their lateral ends bent towards the post-AT linkers with an interdomain angle of 222° 3 (Fig. 1c). The MAS DH dimer is distinct from the V-shaped DH arrangement in FAS 6 , which 4 lacks a dimerization interface and is bent into the opposite direction at an angle of 96°. MAS DH 5 rather resembles linear DH dimers of modPKSs with interdomain angles of 167-203°1 5-17 and a 6 common mode of dimerization via "handshake" interactions between β-strands of the N-terminal 7 hot-dog folds (Extended Data Fig. 1f-h). 8To obtain an authentic representation of the MAS modifying region, we crystallized in 9 presence of NADP + the complete DH-ΨKR-ER-KR segment, which is dimeric in solution based 10 on AUC. Based on SAXS, ACP deletion is not affecting the overall structure of this region 11 (Extended Data Fig. 2a-c). The crystallographic asymmetric unit reveals a complex packing of Fig. 3a, b) (aa 1948-1960) remains disordered in the absence of ligand, and 8 concomitantly, the nicotinamide moiety of NADP + is disordered (Extended Data Fig. 3d). The 9 MAS ΨKR exhibits an N-terminal β-α-β-α extension, which is commonly observed in modPKSs, 10but not in FASs 6,23 ; this extension exhibits increased flexibility as indicated by temperature 11 factor distributions (Extended Data ...

Diguanylate cyclases synthesize the second messenger c-di-GMP, which in turn governs a plethora of physiological processes in bacteria. Although most diguanylate cyclases harbor sensory domains, their input signals are largely unknown. Here, we demonstrate that diguanylate cyclase DgcZ (YdeH) from Escherichia coli is regulated allosterically by zinc. Crystal structures show that the zinc ion is bound to the 3His/1Cys motif of the regulatory chemoreceptor zinc-binding domain, which mediates subunit contact within the dimeric enzyme. In vitro, zinc reversibly inhibits DgcZ with a subfemtomolar Ki constant. In vivo, bacterial biofilm formation is modulated by externally applied zinc in a DgcZ- and c-di-GMP-dependent fashion. The study outlines the structural principles of this zinc sensor. Zinc binding seems to regulate the activity of the catalytic GGDEF domains by impeding their mobility and thus preventing productive encounter of the two GTP substrates.

Cyclic di-GMP (c-di-GMP) is an almost universal bacterial second messenger involved in the regulation of cell surface-associated traits and the persistence of infections. GGDEF and EAL domain-containing proteins catalyse c-di-GMP synthesis and degradation, respectively. We report the enzymatic large-scale synthesis of c-di-GMP, making use of the GGDEF domain-containing protein YdeH from Escherichia coli. Overexpression and purification of YdeH have been established, and the conditions for c-di-GMP synthesis were optimised. In contrast to the chemical synthesis of c-di-GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within 1 day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes.