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Highlights: [•] Cryo-EM structures illuminate human TFIID plasticity. [•] TAF1–TBP crystal structure reveals regulatory roles of TAF1 in transcription. [•] RNAPII–TFIIB initially transcribing complex (ITC) structure details TFIIB function. [•] Crystal structures of the Mediator head module were elucidated. [•] EM studies reveal principles of yeast and human PIC assembly. [ABSTRACT FROM AUTHOR]

The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect ohgomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked ohigomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain. [ABSTRACT FROM AUTHOR]

The only enzyme responsible for cadaverine production in the major multidrug-resistant human pathogen Pseudomonas aeruginosa is the lysine decarboxylase LdcA. This enzyme modulates the general polyamine homeostasis, promotes growth, and reduces bacterial persistence during carbenicillin treatment. Here we present a 3.7-Å resolution cryoelectron microscopy structure of LdcA. We introduce an original approach correlating phylogenetic signal with structural information and reveal possible recombination among LdcA and arginine decarboxylase subfamilies within structural domain boundaries. We show that LdcA is involved in full virulence in an insect pathogenesis model. Furthermore, unlike its enterobacterial counterparts, LdcA is regulated neither by the stringent response alarmone ppGpp nor by the AAA+ ATPase RavA. Instead, the P. aeruginosa ravA gene seems to play a defensive role. Altogether, our study identifies LdcA as an important player in P. aeruginosa physiology and virulence and as a potential drug target. • The structure of the P. aeruginosa LdcA is solved to 3.7-Å resolution by cryo-EM • The phylogenetic signal shifts within structural/functional domains of LAdcs • LdcA is involved in P. aeruginosa physiology and full virulence • P. aeruginosa ravAviaA operon has a potentially defensive function Kandiah et al. solve the structure of the lysine decarboxylase LdcA from a human pathogen Pseudomonas aeruginosa by cryo-EM. They use evolutionary information to analyze the system, demonstrate involvement of LdcA in full virulence of the pathogen in vivo , and propose to target this protein for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Investigates the interaction of substrate with calreticulin, an endoplasmic reticulum chaperone. Thermodynamics of calreticulin-sugar interactions; Calreticulin modeling studies; Discussion of several findings about the recognition of saccharide ligands by calreticulin vis a vis legume lectin, which have the same fold as this chaperone.

Summary Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a “back-to-back” fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling. [ABSTRACT FROM AUTHOR]