Structural analysis of carboxyspermidine dehydrogenase from Helicobacter pylori.

Spermidine is a cationic polyamine that plays key roles in diverse biological processes, including biofilm formation and cell viability in bacteria. In some human gastrointestinal bacteria, such as Helicobacter pylori and Campylobacter jejuni, spermidine is biosynthesized using carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase through an alternative pathway rather than the classical pathway found in most bacteria and eukaryotes. CASDH condenses putrescine and aspartate ß-semialdehyde into carboxyspermidine in an NADPH-dependent manner. Because structural information on CASDH is not available, the exact enzymatic mechanism of CASDH has not been elucidated. To reveal the structural features of CASDH required for cofactor and substrate recruitment, we determined the crystal structures of the H. pylori CASDH protein alone and in complex with NADP. CASDH consists of three domains (D1, D2, and D3) and assembles into a homodimer exclusively using the D3 domain. The CASDH structure harbors a dent between the D1 and D3 domains. The NADP cofactor is inserted into the interdomain dent and induces structural rearrangements in CASDH, including dent closure and local structural changes in the D1 and D3 domains. A comparative analysis suggests that the substrate of CASDH binds in a cavity near the nicotinamide moiety of NADPH for the condensation reaction.

Structural analysis of the Toll-like receptor 15 TIR domain.

Toll-like receptors (TLRs) activate innate immunity in response to pathogen-associated molecular patterns (PAMPs). The ectodomain of a TLR directly senses a PAMP and the intracellular TIR domain dimerizes to initiate a signaling cascade. The TIR domains of TLR6 and TLR10, which belong to the TLR1 subfamily, have been structurally characterized in a dimer, whereas those of other subfamilies, including TLR15, have not been explored at the structural or molecular level. TLR15 is a TLR unique to birds and reptiles that responds to virulence-associated fungal and bacterial proteases. To reveal how the TLR15 TIR domain (TLR15TIR) triggers signaling, the crystal structure of TLR15TIR was determined in a dimeric form and a mutational study was performed. TLR15TIR forms a one-domain structure in which a five-stranded ß-sheet is decorated by α-helices, as shown for TLR1 subfamily members. TLR15TIR exhibits substantial structural differences from other TLRs at the BB and DD loops and αC2 helix that are involved in dimerization. As a result, TLR15TIR is likely to form a dimeric structure that is unique in its intersubunit orientation and the contribution of each dimerizing region. Further comparative analysis of TIR structures and sequences provides insights into the recruitment of a signaling adaptor protein by TLR15TIR.

Activation of the Legionella pneumophila LegK7 Effector Kinase by the Host MOB1 Protein.

Legionella pneumophila infects alveolar macrophages and can cause life-threatening pneumonia in humans. Upon internalization into the host cell, L. pneumophila injects numerous effector proteins into the host cytoplasm as a part of its pathogenesis. LegK7 is an effector kinase of L. pneumophila that functionally mimics the eukaryotic Mst kinase and phosphorylates the host MOB1 protein to exploit the Hippo pathway. To elucidate the LegK7 activation mechanism, we determined the apo structure of LegK7 in an inactive form and performed a comparative analysis of LegK7 structures. LegK7 is a non-RD kinase that contains an activation segment that is ordered, irrespective of stimulation, through a unique ß-hairpin-containing segment, and it does not require phosphorylation of the activation segment for activation. Instead, bacterial LegK7 becomes an active kinase via its heterologous molecular interaction with the host MOB1 protein. MOB1 binding triggers reorientation of the two lobes of the kinase domain, as well as a structural change in the interlobe hinge region in LegK7, consequently reshaping the LegK7 structure into an ATP binding-compatible closed conformation. Furthermore, we reveal that LegK7 is an atypical kinase that contains an N-terminal capping domain and a hydrophilic interlobe linker motif, which play key roles in the MOB1-induced activation of LegK7.