Halogen Bonding in Haspin-Halogenated Tubercidin Complexes: Molecular Dynamics and Quantum Chemical Calculations.

Haspin, an atypical serine/threonine protein kinase, is a potential target for cancer therapy. 5-iodotubercidin (5-iTU), an adenosine derivative, has been identified as a potent Haspin inhibitor in vitro. In this paper, quantum chemical calculations and molecular dynamics (MD) simulations were employed to identify and quantitatively confirm the presence of halogen bonding (XB), specifically halogen∙∙∙π (aromatic) interaction between halogenated tubercidin ligands with Haspin. Consistent with previous theoretical finding, the site specificity of the XB binding over the ortho-carbon is identified in all cases. A systematic increase of the interaction energy down Group 17, based on both quantum chemical and MD results, supports the important role of halogen bonding in this series of inhibitors. The observed trend is consistent with the experimental observation of the trend of activity within the halogenated tubercidin ligands (F < Cl < Br < I). Furthermore, non-covalent interaction (NCI) plots show that cooperative non-covalent interactions, namely, hydrogen and halogen bonds, contribute to the binding of tubercidin ligands toward Haspin. The understanding of the role of halogen bonding interaction in the ligand-protein complexes may shed light on rational design of potent ligands in the future.

A Novel Missense Mutation in TNNI3K Causes Recessively Inherited Cardiac Conduction Disease in a Consanguineous Pakistani Family.

Cardiac conduction disease (CCD), which causes altered electrical impulse propagation in the heart, is a life-threatening condition with high morbidity and mortality. It exhibits genetic and clinical heterogeneity with diverse pathomechanisms, but in most cases, it disrupts the synchronous activity of impulse-generating nodes and impulse-conduction underlying the normal heartbeat. In this study, we investigated a consanguineous Pakistani family comprised of four patients with CCD. We applied whole exome sequencing (WES) and co-segregation analysis, which identified a novel homozygous missense mutation (c.1531T>C;(p.Ser511Pro)) in the highly conserved kinase domain of the cardiac troponin I-interacting kinase (TNNI3K) encoding gene. The behaviors of mutant and native TNNI3K were compared by performing all-atom long-term molecular dynamics simulations, which revealed changes at the protein surface and in the hydrogen bond network. Furthermore, intra and intermolecular interaction analyses revealed that p.Ser511Pro causes structural variation in the ATP-binding pocket and the homodimer interface. These findings suggest p.Ser511Pro to be a pathogenic variant. Our study provides insights into how the variant perturbs the TNNI3K structure-function relationship, leading to a disease state. This is the first report of a recessive mutation in TNNI3K and the first mutation in this gene identified in the Pakistani population.

When is an obscurin variant pathogenic? The impact of Arg4344Gln and Arg4444Trp variants on protein-protein interactions and protein stability.

Obscurin is a giant muscle protein that connects the sarcomere with the sarcoplasmic reticulum, and has poorly understood structural and signalling functions. Increasingly, obscurin variants are implicated in the pathophysiology of cardiovascular diseases. The Arg4344Gln variant (R4344Q) in obscurin domain Ig58, initially discovered in a patient with hypertrophic cardiomyopathy, has been reported to reduce binding to titin domains Z8-Z9, impairing obscurin's Z-disc localization. An R4344Q knock-in mouse developed a cardiomyopathy-like phenotype with abnormal Ca2+-handling and arrhythmias, which were attributed to an enhanced affinity of a putative interaction between obscurin Ig58 and phospholamban (PLN) due to the R4344Q variant. However, the R4344Q variant is found in 15% of African Americans, arguing against its pathogenicity. To resolve this apparent paradox, we quantified the influence of the R4344Q variant (alongside another potentially pathogenic variant: Arg4444Trp (R4444W)) on binding to titin Z8-Z9, novex-3 and PLN using pull-down assays and microscale thermophoresis and characterized the influence on domain stability using differential scanning fluorimetry. We found no changes in titin binding and thermostability for both variants and modestly increased affinities of PLN for R4344Q and R4444W. While we could not confirm the novex-3/obscurin interaction, the PLN/obscurin interaction relies on the transmembrane region of PLN and is not reproducible in mammalian cells, suggesting it is an in vitro artefact. Without clear clinical evidence for disease involvement, we advise against classifying these obscurin variants as pathogenic.

Structural basis for the final steps of human 40S ribosome maturation.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins1,2. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB13. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO14. The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae.

InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1-SMG8-SMG9 complex.

We report the 3.45-Å resolution cryo-EM structure of human SMG1-SMG8-SMG9, a phosphatidylinositol-3-kinase (PI(3)K)-related protein kinase (PIKK) complex central to messenger RNA surveillance. Structural and MS analyses reveal the presence of inositol hexaphosphate (InsP6) in the SMG1 kinase. We show that the InsP6-binding site is conserved in mammalian target of rapamycin (mTOR) and potentially other PIKK members, and that it is required for optimal in vitro phosphorylation of both SMG1 and mTOR substrates.

The long-awaited structure of HIPK2.

Homeodomain-interacting protein kinases (HIPKs) are kinases that phosphorylate transcription factors involved in cell proliferation, differentiation, and apoptosis. Their structures have been long sought because of their potential as drug targets in cancers and fibrosis. Agnew and colleagues present the first crystal structure of the HIPK2 kinase domain, complexed with the small-molecule inhibitor CX-4945, revealing important structural differences from related protein kinases of the DYRK family. This structure provides a starting point to exploit HIPK2's distinct structural features to develop selective small-molecule inhibitors of this kinase.

The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation.

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.

Tailing miniSOG: structural bases of the complex photophysics of a flavin-binding singlet oxygen photosensitizing protein.

miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.

Structure of a eukaryotic cytoplasmic pre-40S ribosomal subunit.

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.

Impact of intracellular ionic strength on dimer binding in the NF-kB Inducing kinase.

Improper signaling of the nuclear factor-κB (NF-κB) pathway plays a critical role in many inflammatory disease states including cancer, stroke, and viral infections. Although the signaling pathways are known, how these molecular mechanisms respond to changes in the intracellular microenvironment such as pH, ionic strength, and temperature, remains elusive. Molecular dynamics simulations were employed to differentiate the structural dynamics of the NF-κB Inducing Kinase (NIK), a protein kinase responsible for invoking the non-canonical NF-κB pathway, in its native and mutant form, and in the absence and presence of salt concentration in efforts to probe whether changes in the ionic environment stabilize or destabilize the NIK dimer. Analyses of structure-activity and conformational-activity relationships indicate that the protein-protein interactions are sensitive to changes in the ionic strength. Ligand binding pockets as well as regions between the oligomer interface either compress or expand, affecting both local and distal intermolecular interactions that result in stabilization or destabilization in the protein assembly.

3.9 Å structure of the yeast Mec1-Ddc2 complex, a homolog of human ATR-ATRIP.

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master regulator of DNA damage response and replication stress in humans, but the mechanism of its activation remains unclear. ATR acts together with its partner ATRIP. Using cryo-electron microscopy, we determined the structure of intact Mec1-Ddc2 (the yeast homolog of ATR-ATRIP), which is poised for catalysis, at a resolution of 3.9 angstroms. Mec1-Ddc2 forms a dimer of heterodimers through the PRD and FAT domains of Mec1 and the coiled-coil domain of Ddc2. The PRD and Bridge domains in Mec1 constitute critical regulatory sites. The activation loop of Mec1 is inhibited by the PRD, revealing an allosteric mechanism of kinase activation. Our study clarifies the architecture of ATR-ATRIP and provides a structural framework for the understanding of ATR regulation.

Human GRP78 affinity towards its signaling partners Ire1α and PERK is differently modulated by an unfolded protein client.

Protein-folding stress is characteristic of specialized secretory cells and plays a dominant role in a multitude of diseases. The unfolded protein response (UPR) thus triggered is a proteostatic signaling network that adapts the protein-folding capacity of the endoplasmic reticulum to the cellular demands. We have measured the binding affinities between human GRP78, an essential chaperone located in ER, and two transmembrane UPR sensors (human PERK and Ire1α), with or without the addition of an unfolded protein client. We reveal distinct binding affinities between the binary and ternary complexes thus formed, that suggest a preference for the PERK signaling branch under stress, and a predilection for the GRP78-UPR sensor complex formation upon stressor removal. These results imply a gated UPR mechanism that tunes the overall cellular behavior to the accumulation of unfolded proteins.

Molecular basis of APC/C regulation by the spindle assembly checkpoint.

In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/C­MCC complex (APC/C(MCC)). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20(MCC)) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced.

Organization of TNIK in dendritic spines.

Tumor necrosis factor receptor-associated factor 2 (TRAF2)- and noncatalytic region of tyrosine kinase (NCK)-interacting kinase (TNIK) has been identified as an interactor in the psychiatric risk factor, Disrupted in Schizophrenia 1 (DISC1). As a step toward deciphering its function in the brain, we performed high-resolution light and electron microscopic immunocytochemistry. We demonstrate here that TNIK is expressed in neurons throughout the adult mouse brain. In striatum and cerebral cortex, TNIK concentrates in dendritic spines, especially in the vicinity of the lateral edge of the synapse. Thus, TNIK is highly enriched at a microdomain critical for glutamatergic signaling.

Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.

Mutations in SNF1 complex genes affect yeast cell wall strength.

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.

Single-molecule experiments reveal the flexibility of a Per-ARNT-Sim domain and the kinetic partitioning in the unfolding pathway under force.

Per-ARNT-Sim (PAS) domains serve as versatile binding motifs in many signal-transduction proteins and are able to respond to a wide spectrum of chemical or physical signals. Despite their diverse functions, PAS domains share a conserved structure. It has been suggested that the structure of PAS domains is flexible and thus adaptable to many binding partners. However, direct measurement of the flexibility of PAS domains has not yet been provided. Here, we quantitatively measure the mechanical unfolding of a PAS domain, ARNT PAS-B, using single-molecule atomic force microscopy. Our force spectroscopy results indicate that the structure of ARNT PAS-B can be unraveled under mechanical forces as low as ~30 pN due to its broad potential well for the mechanical unfolding transition of ~2 nm. This allows the PAS-B domain to extend by up to 75% of its resting end-to-end distance without unfolding. Moreover, we found that the ARNT PAS-B domain unfolds in two distinct pathways via a kinetic partitioning mechanism. Sixty-seven percent of ARNT PAS-B unfolds through a simple two-state pathway, whereas the other 33% unfolds with a well-defined intermediate state in which the C-terminal ß-hairpin is detached. We propose that the structural flexibility and force-induced partial unfolding of PAS-B domains may provide a unique mechanism for them to recruit diverse binding partners and lower the free-energy barrier for the formation of the binding interface.

Molecular architecture of the "stressosome," a signal integration and transduction hub.

A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium-resolution cryo-electron microscopy reconstruction of the 1.8-megadalton "stressosome" from Bacillus subtilis. Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid-like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult.

Predikin and PredikinDB: a computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites.

BACKGROUND: We have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript. RESULTS: Here, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i) PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii) a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC) graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features. CONCLUSION: New features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at http://predikin.biosci.uq.edu.au.

Fluorescence resonance energy transfer in quantum dot-protein kinase assemblies.

In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His(6)-PDK1(DeltaPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with the His(6)-PDK1(DeltaPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of the His(6)-PDK1(DeltaPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking the His(6)-PDK1(DeltaPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles.