Cooperative regulation of C1-domain membrane recruitment polarizes atypical Protein Kinase C

Recruitment of the Par complex protein atypical Protein Kinase C (aPKC) to a specific membrane domain is a key step in the polarization of animal cells. While numerous proteins and phospholipids interact with aPKC, how these interactions cooperate to control its membrane recruitment has been unknown. Here we identify aPKC's C1 domain as a phospholipid interaction module that targets aPKC to the membrane of Drosophila neural stem cells (NSCs). The isolated C1 binds the NSC membrane in an unpolarized manner during interphase and mitosis and is uniquely sufficient among aPKC domains for targeting. Other domains, including the catalytic module and those that bind the upstream regulators Par-6 and Baz, restrict C1's membrane targeting activity spatially and temporally-to the apical NSC membrane during mitosis. Our results suggest that Par complex polarity results from cooperative activation of autoinhibited C1 membrane binding activity.

Structural anatomy of C1 domain interactions with DAG and other agonists

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. DAG effector proteins compete for available lipid using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the first high-resolution crystal structures of a C1 domain (C1B from PKCdelta) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins.

Computational Screening of Phytochemicals against Munc13-1, a Promising target to treat Alcoholism

In silico analysis and characterization has revolutionized target and drug discovery significantly. Alcohol abuse is a big threat to society, economy and wellbeing of people. It has increased the overall disease and injury burden, globally. Recently, a study revealed a brain protein, Munc13-1 C1 domain to play a significant role in development of alcohol tolerance, by binding to alcohol molecules, eventually leading to Alcohol Use Disorder. The aim of this study was to discover a phytochemical that would attach to our target protein, Munc13-1 C1 domain so that it cannot bind with the alcohol molecules. Munc13-1 3D structure obtained from PDB was docked against a library of compounds by MOE software. Ten phytochemicals based on their binding affinity with the target protein were shortlisted i.e. Tannic Acid, Anemone blue anthocyanin 1, Oolonghomobisflavan B, Diosmin, Oolonghomobisflavan A, Neodiosmin, Blepharocalyxin B, 8-Hydroxyhesperetin, Eupatorin and Monotesone A. However, only 8-Hydroxyhesperetin, Eupatorin and Monotesone A followed Lipinski rules. They were non-toxic and non-carcinogenic according to SwissADME. Moreover, have a good drug-like model score as analysed by Molsoft. Further, in-vivo and invitro examinations are required to inspect their role in reducing alcohol tolerance.

Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd2+ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd2+ spontaneously replaced Zn2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd2+ substitution for Zn2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys3His metal-ion sites of C1B have higher affinity to Cd2+ than Zn2+, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd2+. Our results indicate that Cd2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.

Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

Ring-shaped multimeric structure enables the acceleration of KaiB-KaiC complex formation induced by the ADP/ATP exchange inhibition

Circadian clocks tick a rhythm with a nearly 24-hour period in various organisms. The clock proteins of cyanobacteria, KaiA, KaiB, and KaiC, compose a minimum circadian clock. The slow KaiB-KaiC complex formation, which is essential in determining the clock period, occurs when the C1 domain of KaiC binds ADP produced by ATP hydrolysis. KaiC is considered to promote this complex formation by inhibiting the backward process, ADP/ATP exchange, rather than activating the forward process, ATP hydrolysis. Remarkably, although inhibition of backward process, in general, decelerates the whole process, KaiC oppositely accelerates the complex formation. In this article, by building a novel reaction model, we investigate the molecular mechanism of the simultaneous promotion and acceleration of the complex formation, which may play a significant role in keeping the period invariant under environmental perturbations. Based on several experimental results, we assume in this model that six KaiB monomers cooperatively and rapidly binds to C1 with the stabilization of the binding-competent conformation of C1 only when C1 binds six ADP. We find the cooperative KaiB binding effectively separates the pre-binding process of C1 into a fast transformation to binding-competent C1 requiring multiple ATP hydrolyses and its slow backward transformation. Since the ADP/ATP exchange retards the forward process, its inhibition results in the acceleration of the complex formation. We also find that, in a simplified monomeric model where KaiB binds to a KaiC monomer independently of the other monomers, the ADP/ATP exchange inhibition cannot accelerate the complex formation. In summary, we conclude that the ring-shaped hexameric form of KaiC enables the acceleration of the complex formation induced by the backward process inhibition because the cooperative KaiB binding arises from the structure of KaiC.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Structure of Blood Coagulation Factor VIII in Complex with an Anti-C1 Domain Pathogenic Antibody Inhibitor

Antibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a Group A anti-C1 domain inhibitor, termed 2A9, in complex with a B domain-deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at three different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156 and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor (vWF). Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti-C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.

Design, Synthesis and Characterization of Novel sn-1 Heterocyclic DAGlactones as PKCe Activators

<p>In this study we describe the synthesis and characterization of novel diacylglycerol (DAG)-lactones that bind to protein kinase C (PKC). DAG-lactones proved to be useful templates for the design of potent and selective C1 domain ligands. The ester moiety at <i>sn-1</i> position, a common feature in this template, is relevant for interaction with the PKC C1 domains, although it represents a labile group susceptible to endogenous esterases. Our studies identified the DAG-lactone 10B12 with an isozazole ring as a nanomolar affinity PKC ligand. This compound shows preferential selectivity for PKCepsilon, and strongly activates actin cytoskeleton reorganization into peripheral ruffles in cancer cells, an effect mediated by PKCepsilon. Therefore, introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC selectivity.</p><div><br></div>

Design, Synthesis and Characterization of Novel sn-1 Heterocyclic DAGlactones as PKCe Activators

<p>In this study we describe the synthesis and characterization of novel diacylglycerol (DAG)-lactones that bind to protein kinase C (PKC). DAG-lactones proved to be useful templates for the design of potent and selective C1 domain ligands. The ester moiety at <i>sn-1</i> position, a common feature in this template, is relevant for interaction with the PKC C1 domains, although it represents a labile group susceptible to endogenous esterases. Our studies identified the DAG-lactone 10B12 with an isozazole ring as a nanomolar affinity PKC ligand. This compound shows preferential selectivity for PKCepsilon, and strongly activates actin cytoskeleton reorganization into peripheral ruffles in cancer cells, an effect mediated by PKCepsilon. Therefore, introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC selectivity.</p><div><br></div>