The mitochondrial Lon protease (LonP1) regulates mitochondrial health by removing redundant proteins from the mitochondrial matrix. We determined LonP1 in eight nucleotide-dependent conformational states by cryoelectron microscopy (cryo-EM). The flexible assembly of N-terminal domains had 3-fold symmetry, and its orientation depended on the conformational state. We show that a conserved structural motif around T803 with a high similarity to the trypsin catalytic triad is essential for proteolysis. We show that LonP1 is not regulated by redox potential, despite the presence of two conserved cysteines at disulfide-bonding distance in its unfoldase core. Our data indicate how sequential ATP hydrolysis controls substrate protein translocation in a 6-fold binding change mechanism. Substrate protein translocation, rather than ATP hydrolysis, is a rate-limiting step, suggesting that LonP1 is a Brownian ratchet with ATP hydrolysis preventing translocation reversal. 3-fold rocking motions of the flexible N-domain assembly may assist thermal unfolding of the substrate protein.

DNA end resection mediated by the coordinated action of nuclease and helicase is a crucial step in initiating homologous recombination. The end-resection apparatus NurA nuclease and HerA helicase are present in both archaea and bacteria. Here, we report the cryo-electron microscopy structure of a bacterial HerA-NurA complex from Deinococcus radiodurans. The structure reveals a barrel-like hexameric HerA and a distinctive NurA dimer subcomplex, which has a unique extended N-terminal region (ENR) involved in bacterial NurA dimerization and activation. In addition to the long protruding linking loop and the C-terminal α helix of NurA, the flexible ENR is close to the HerA-NurA interface and divides the central channel of the DrNurA dimer into two halves, suggesting a possible mechanism of DNA end processing. In summary, this work provides new insights into the structure, assembly, and activation mechanisms of bacterial DNA end resection mediated by a minimal end-resection apparatus.

Comment on Structure. 2022 Sep 1;30(9):1245-1253.e5.

Comment in Structure. 2022 Sep 1;30(9):1219-1221.

Comment in Structure. 2022 Sep 1;30(9):1221-1223.

The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KSQ, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KSQ and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KSQ and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KSQ active site, while an AT-swapped version of Pik127 demonstrated that KSQ can also decarboxylate malonyl groups. Mechanisms for how KSQ and KS domains catalyze carbon-carbon chemistry are proposed.

Comment on Structure. 2022 Sep 1;30(9):1269-1284.e6.

The solute carrier (SLC) superfamily is the largest group of proteins responsible for the transmembrane transport of substances in human cells. It includes more than 400 members that are organized into 65 families according to their physiological function and sequence similarity. Different families of SLCs can adopt the same or different folds that determine the mechanism and reflect the evolutionary relationship between SLC members. Analysis of structural data in the literature before this work showed 13 different folds in the SLC superfamily covering 40 families and 343 members. To further study their mechanism, we systematically explored the SLC superfamily to look for more folds. Based on our results, at least three new folds are found for the SLC superfamily, one of which is in the choline-like transporter family (SLC44) and has been experimentally verified. Our work has laid a foundation and provided important insights for the systematic and comprehensive study of the structure and function of SLC.

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

The mitochondrial serine protease High-temperature requirement A2 (HtrA2) is associated with various diseases including neurodegenerative disorders and cancer. Despite availability of structural details, the reports on HtrA2's mechanistic regulation that varies with the type of activation signals still remain non-concordant. To expound the role of regulatory PDZ (Postsynaptic density-95/Discs large/Zonula occludens-1) domains in multimodal activation of HtrA2, we generated heterotrimeric HtrA2 variants comprising different numbers of PDZs and/or active-site mutations. Sequential deletion of PDZs from the trimeric ensemble significantly affected its residual activity in a way that proffered a hypothesis advocating inter-molecular allosteric crosstalk via PDZs in HtrA2. Furthermore, structural and computational snapshots affirmed the role of PDZs in secondary structural element formation around the regulatory loops and coordinated reorganization of the N-terminal region. Therefore, apart from providing cues for devising structure-guided therapeutic strategies, this study establishes a physiologically relevant working model of complex allosteric regulation through a trans-mediated cooperatively shared energy landscape.

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their "pocket domain" to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity.

Update of bioRxiv. 2022 Jan 26;:

Virulence in Pseudomonas aeruginosa (PA) depends on complex regulatory networks, involving phosphorelay systems based on two-component systems (TCSs). The GacS/GacA TCS is a master regulator of biofilm formation, swarming motility, and virulence. GacS is a membrane-associated unorthodox histidine kinase (HK) whose phosphorelay signaling pathway is inhibited by the RetS hybrid HK. Here we provide structural and functional insights into the interaction of GacS with RetS. The structure of the GacS-HAMP-H1 cytoplasmic regions reveals an unusually elongated homodimer marked by a 135 Å long helical bundle formed by the HAMP, the signaling helix (S helix) and the DHp subdomain. The HAMP and S helix regions are essential for GacS signaling and contribute to the GacS/RetS binding interface. The structure of the GacS D1 domain together with the discovery of an unidentified functional ND domain, essential for GacS full autokinase activity, unveils signature motifs in GacS required for its atypical autokinase mechanism.