Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptormediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered. Here, structures of phosphorylated and unphosphorylated human TBK1 kinase and ubiquitin-like domains, combined with biochemical studies, indicate a molecular mechanism of activation via transautophosphorylation. These TBK1 structures are consistent with the tripartite architecture observed recently for the related kinase IKKβ, but domain contributions toward target recognition appear to differ for the two enzymes. In particular, both TBK1 autoactivation and substrate specificity are likely driven by signal-dependent colocalization events. Phosphorylation promotes the dimerization and nuclear translocation of these transcription factors that stimulate production of type I interferons (IFNs) (1,3,5). Recent studies have identified an additional role for TBK1 in the xenophagic elimination of bacteria (6-9) and better-defined how cross-talk within the IKK family regulates innate immune response (10).Under pathological conditions, IKK-mediated pathways can also be activated inappropriately by endogenous signals, contributing to inflammatory disorders and oncogenesis (11,12). Whereas canonical IKKs have long been recognized as bridges between chronic inflammation and cancer, IKK-related kinases more recently have also been implicated in cell transformation and tumor progression (13). TBK1 has been of particular interest, given its identification both as an activator of the oncogenic AKT kinase (14-18) and as an essential factor in KRAS-driven cancers (19).TBK1 activity is regulated by phosphorylation on S172 within the classical kinase activation loop. Serine-to-alanine substitution at this position abolishes TBK1 activity, whereas the phosphomimetic mutation S172E partially restores activity to within ∼200-fold of the wild-type kinase (20). Genetic and pharmacological inhibition studies have indicated that TBK1 can be activated by IKKβ, as well as by apparent autophosphorylation (10). Additional posttranslational modifications of TBK1 lysine residues by K63-linked polyubiquitin chains have been shown to promote production of IFNs in viral infections (21).TBK1 contains a predicted ubiquitin-like domain (ULD) (22) that is located between the N-terminal kinase domain (KD) and the C-terminal scaffolding/dimerization domain (SDD), a domain arrangement that appears to be shared among the IKK family of kinases (3). Deletion or mutation of the ULD in TBK1 or IKKε severely impairs kinase activation and substrate phosphorylation in cells (22,23). Furthermore, the integrity of the ULD in IKKβ is not only required for kinase activity (24) bu...

α-Helical antimicrobial peptides (AMPs) generally have facially amphiphilic structures that may lead to undesired peptide interactions with blood proteins and self-aggregation due to exposed hydrophobic surfaces. Here we report the design of a class of cationic, helical homo-polypeptide antimicrobials with a hydrophobic internal helical core and a charged exterior shell, possessing unprecedented radial amphiphilicity. The radially amphiphilic structure enables the polypeptide to bind effectively to the negatively charged bacterial surface and exhibit high antimicrobial activity against both gram-positive and gram-negative bacteria. Moreover, the shielding of the hydrophobic core by the charged exterior shell decreases nonspecific interactions with eukaryotic cells, as evidenced by low hemolytic activity, and protects the polypeptide backbone from proteolytic degradation. The radially amphiphilic polypeptides can also be used as effective adjuvants, allowing improved permeation of commercial antibiotics in bacteria and enhanced antimicrobial activity by one to two orders of magnitude. Designing AMPs bearing this unprecedented, unique radially amphiphilic structure represents an alternative direction of AMP development; radially amphiphilic polypeptides may become a general platform for developing AMPs to treat drug-resistant bacteria.

Antimicrobial peptides (AMPs) are a diverse class of well-studied membrane-permeating peptides with important functions in innate host defense. In this short review, we provide a historical overview of AMPs, summarize previous applications of machine learning to AMPs, and discuss the results of our studies in the context of the latest AMP literature. Much work has been recently done in leveraging computational tools to design new AMP candidates with high therapeutic efficacies for drug-resistant infections. We show that machine learning on AMPs can be used to identify essential physico-chemical determinants of AMP functionality, and identify and design peptide sequences to generate membrane curvature. In a broader scope, we discuss the implications of our findings for the discovery of membrane-active peptides in general, and uncovering membrane activity in new and existing peptide taxonomies.

The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magicangle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31 P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1 H-31 P 2D correlation spectra and 2 H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation.solid-state NMR spectroscopy | small-angle X-ray scattering | conformational polymorphism | membrane curvature | peptide-membrane interactions V iral fusion proteins mediate entry of enveloped viruses into cells by merging the viral lipid envelope and the cell membrane. The membrane-interacting subunit of these glycoproteins contains two hydrophobic domains: a fusion peptide (FP) that is usually located at the N terminus and a transmembrane domain (TMD) at the C terminus (1). Together, these domains sandwich a water-soluble ectodomain with a helical segment that trimerizes into a coiled coil. During virus-cell fusion, the trimeric protein, which initially adopts a compact structure, unfolds to an extended intermediate that exposes the FP to the target cell membrane while keeping the TMD in the virus envelope. This extended conformation then folds onto itself to form a trimer of hairpins, in so doing pulling the cell membrane and the virus envelope into close proximity (2, 3). Subsequently, the FP and TMD are hypothesized to deform the two membranes and dehydrate them (4), eventually causing a fusion pore and a fully merged membrane. In the postfusion state, most viral fusion proteins exhibit a six-helixbundle structure in the ectodomain due to the trimer of hairpins (5).This model of virus-cell fusion largely derives from crystal structures of fusion proteins in the pre-and postfusion st...