Molecular interaction between natural IgG and ficolin - Mechanistic insights on adaptive-innate immune crosstalk

10.1038/srep03675Scientific Reports

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1

Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem(GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins

SUMO Suppresses the Activity of the Jasmonic Acid Receptor CORONATINE INSENSITIVE1

Plants respond rapidly to sudden environmental cues, often responding prior to changes in the hormone levels that coordinate these responses. How this is achieved is not fully understood. The integrative role of the phytohormone jasmonic acid (JA) relies upon the plant's ability to control the levels of JASMONATE ZIM (JAZ) domain-containing repressor proteins. Here, we demonstrate that regardless of intrinsic JA levels, Small Ubiquitin-like Modifier (SUMO)-conjugated JAZ proteins inhibit the JA receptor CORONATINE INSENSITIVE1 (COI1) from mediating non-SUMOylated JAZ degradation. The SUMO-deconjugating proteases OVERLY TOLERANT TO SALT1 (OTS1) and OTS2 regulate JAZ protein SUMOylation and stability. The ots1 ots2 double mutants accumulate SUMOylated and non-SUMOylated JAZ repressor proteins but show no change in endogenous JA levels compared with wild-type plants. SUMO1-conjugated JAZ proteins bind to COI1 independently of the JA mimic coronatine. SUMO inhibits JAZ binding to COI1. We identify the SUMO interacting motif in COI1 and demonstrate that this is vital to SUMO-dependent inhibition of COI1. Necrotroph infection of Arabidopsis thaliana promotes SUMO protease degradation, and this increases JAZ SUMOylation and abundance, which in turn inhibits JA signaling. This study reveals a mechanism for rapidly regulating JA responses, allowing plants to adapt to environmental changes

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.Published versio

Secreted M-ficolin anchors onto monocyte transmembrane G protein-coupled receptor 43 and cross talks with plasma C-reactive protein to mediate immune signaling and regulate host defense

10.4049/jimmunol.1001225Journal of Immunology185116899-6910JOIM

Human antibody C10 neutralizes by diminishing Zika but enhancing dengue virus dynamics

10.1016/j.cell.2021.11.00

HDX-MS of RshA.

<p>(<b>A</b>) Sequence coverage map for RshA. Solid line denotes the peptic fragments analyzed in the study with total sequence coverage of 88%. (<b>B</b>) ESI-Q-TOF mass spectra for one pepsin digest fragment of RshA (35–57) m/z = 881.084, z = 3, which showed significant difference upon RshA binding. (i) Undeuterated RshA peptide (ii) The isotopic envelop for the same peptide from free RshA following 10min deuteration; (iii) The isotopic envelope for the same peptide from RshA and SigH complex following 10 min deuteration., The isotopic envelope for the same peptide. (<b>C</b>) The protein is shown in magenta. The region in red represents regions showing decreased exchange upon interactions with its partner, SigH.</p

Summary of amide H/D exchange in pepsin digest fragments from SigH (Deuterium exchange time = 10 min)^a.

a<p>Average number of deuterons exchanged determined following 10-min deuterium exchange. Values reported are the mean and standard deviation from at least two independent experiments.</p