Ezrin interacts with S100A4 via both its N- and C-terminal domains
PLoS One. 2017 May 11;12(5):e0177489
Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process.
Consensus Prediction of Charged Single Alpha-Helices with CSAHserver
Methods Mol Biol. 2017;1484:25-34
Charged single alpha-helices (CSAHs) constitute a rare structural motif. CSAH is characterized by a high density of regularly alternating residues with positively and negatively charged side chains. Such segments exhibit unique structural properties; however, there are only a handful of proteins where its existence is experimentally verified. Therefore, establishing a pipeline that is capable of predicting the presence of CSAH segments with a low false positive rate is of considerable importance. Here we describe a consensus-based approach that relies on two conceptually different CSAH detection methods and a final filter based on the estimated helix-forming capabilities of the segments. This pipeline was shown to be capable of identifying previously uncharacterized CSAH segments that could be verified experimentally. The method is available as a web server at http://csahserver.itk.ppke.hu and also a downloadable standalone program suitable to scan larger sequence collections.
Bioinformatics Approaches to the Structure and Function of Intrinsically Disordered Proteins
J. Rigden D. (eds) From Protein Structure to Function with Bioinformatics pp 167-203. Springer, Dordrecht; ISBN 978-94-024-1069-3; DOI:10.1007/978-94-024-1069-3_6
Intrinsically disordered proteins and protein regions (IDPs/IDRs) exist without a well-defined structure. They carry out their function by relying on their highly flexible conformational states and are mostly involved in signal transduction and regulation. By a battery of biophysical techniques, the structural disorder of about 600 proteins has been demonstrated, and functional studies have provided the basis of classifying their functions into various schemes. Indirect evidence suggests that the occurrence of disorder is widespread, and several thousand proteins with significant disorder exist in the human proteome alone. To narrow the wide gap between known and anticipated IDPs, a range of bioinformatics algorithms have been developed, which can reliably predict the disordered state from the amino acid sequence. Attempts have also been made to predict IDP function. However, due to their fast evolution, and reliance on short motifs for function, capturing sequence clues for IDP functions is a much more challenging task. In this chapter we give a brief survey of the IDP field, with particular focus on their functions and bioinformatics approaches developed for predicting their structure and function.
DIBS: a repository of disordered binding sites mediating interactions with ordered proteins
Bioinformatics, btx640, https://doi.org/10.1093/bioinformatics/btx640
Intrinsically Disordered Proteins (IDPs) mediate crucial protein–protein interactions, most notably in signaling and regulation. As their importance is increasingly recognized, the detailed analyses of specific IDP interactions opened up new opportunities for therapeutic targeting. Yet, large scale information about IDP-mediated interactions in structural and functional details are lacking, hindering the understanding of the mechanisms underlying this distinct binding mode.
Here, we present DIBS, the first comprehensive, curated collection of complexes between IDPs and ordered proteins. DIBS not only describes by far the highest number of cases, it also provides the dissociation constants of their interactions, as well as the description of potential post-translational modifications modulating the binding strength and linear motifs involved in the binding. Together with the wide range of structural and functional annotations, DIBS will provide the cornerstone for structural and functional studies of IDP complexes.
Availability and implementation
DIBS is freely accessible at http://dibs.enzim.ttk.mta.hu/. The DIBS application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created.
Dynamic changes in binding interaction networks of sex steroids establish their non-classical effects
Sci Rep. 2017 Nov 1;7(1):14847
Non-classical signaling in the intracellular second messenger system plays a pivotal role in the cytoprotective effect of estradiol. Estrogen receptor is a common target of sex steroids and important in mediating estradiol-induced neuroprotection. Whereas the mechanism of genomic effects of sex steroids is fairly understood, their non-classical effects have not been elucidated completely. We use real time molecular dynamics calculations to uncover the interaction network of estradiol and activator estren. Besides steroid interactions, we also investigate the co-activation of the receptor. We show how steroid binding to the alternative binding site of the non-classical action is facilitated by the presence of a steroid in the classical binding site and the absence of the co-activator peptide. Uncovering such dynamic mechanisms behind steroid action will help the structure-based design of new drugs with non-classical responses and cytoprotective potential.
Maternal alterations in the proteome of the medial prefrontal cortex in rat
J Proteomics. 2017 Feb 5;153:65-77
Proteomic differences between rat dams and control mothers deprived of their pups immediately after delivery were investigated in the medial prefrontal cortex (mPFC). A 2-D DIGE minimal dye technique combined with LC-MS/MS identified 32 different proteins that showed significant changes in expression in the mPFC, of which, 25 were upregulated and 7 were downregulated in dams. The identity of one significantly increased protein, the small heat-shock protein alpha-crystallin B chain (Cryab), was confirmed via Western blot analysis. Alpha-crystallin B chain was distributed in scattered cells in the mPFC, as demonstrated by immunohistochemistry. Furthermore, it was found to be localized in parvalbumin-containing neurons using double labeling. The elevation of its mRNA level in rat dams was also demonstrated via RT-PCR. The functional classification of the altered proteins was conducted using the UniProt and Gene Ontology protein databases. The identified proteins predominantly participate in synaptic transport and plasticity, neuron development, oxidative stress and apoptosis, and cytoskeleton organization. A common regulator and target analysis of these proteins determined using the Elsevier Pathway Studio Platform suggests that protein level changes associated with pup nursing are driven by growth factors and cytokines, while the MAP kinase pathway was identified as a common target. A high proportion of the proteins that were found to be altered in the mPFC are associated with depression.
InterPro in 2017-beyond protein family and domain annotations
Nucleic Acids Res. 2017 Jan 4;45(D1):D190-D199
InterPro (http://www.ebi.ac.uk/interpro/) is a freely available database used to classify protein sequences into families and to predict the presence of important domains and sites. InterProScan is the underlying software that allows both protein and nucleic acid sequences to be searched against InterPro's predictive models, which are provided by its member databases. Here, we report recent developments with InterPro and its associated software, including the addition of two new databases (SFLD and CDD), and the functionality to include residue-level annotation and prediction of intrinsic disorder. These developments enrich the annotations provided by InterPro, increase the overall number of residues annotated and allow more specific functional inferences.
DisProt 7.0: a major update of the database of disordered proteins
Nucleic Acids Res. 2017 Jan 4;45(D1):D219-D227
The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.
Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes
J Biol Chem. 2017 Feb 17;292(7):2690-2702
Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.
Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination
Proc Natl Acad Sci U S A. 2017 Jan 24;114(4):E466-E475
Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.
The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses
Mol Cell Neurosci. 2017 Mar;79:64-80
Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.
Degrons in cancer
Sci Signal. 2017 Mar 14;10(470)
Degrons are the elements that are used by E3 ubiquitin ligases to target proteins for degradation. Most degrons are short linear motifs embedded within the sequences of modular proteins. As regulatory sites for protein abundance, they are important for many different cellular processes, such as progression through the cell cycle and monitoring cellular hypoxia. Degrons enable the elimination of proteins that are no longer required, preventing their possible dysfunction. Although the human genome encodes ~600 E3 ubiquitin ligases, only a fraction of these enzymes have well-defined target degrons. Thus, for most cellular proteins, the destruction mechanisms are poorly understood. This is important for many diseases, especially for cancer, a disease that involves the enhanced expression of oncogenes and the persistence of encoded oncoproteins coupled with reduced abundance of tumor suppressors. Loss-of-function mutations occur in the degrons of several oncoproteins, such as the transcription factors MYC and NRF2, and in various mitogenic receptors, such as NOTCH1 and several receptor tyrosine kinases. Mutations eliminating the function of the β-catenin degron are found in many cancers and are considered one of the most abundant mutations driving carcinogenesis. In this Review, we describe the current knowledge of degrons in cancer and suggest that increased research on the "dark degrome" (unknown degron-E3 relationships) would enhance progress in cancer research.
Simultaneous quantification of protein order and disorder
Nat Chem Biol. 2017 Mar 22;13(4):339-342
Nuclear magnetic resonance spectroscopy is transforming our views of proteins by revealing how their structures and dynamics are closely intertwined to underlie their functions and interactions. Compelling representations of proteins as statistical ensembles are uncovering the presence and biological relevance of conformationally heterogeneous states, thus gradually making it possible to go beyond the dichotomy between order and disorder through more quantitative descriptions that span the continuum between them.
Interaction of mycotoxin zearalenone with human serum albumin
J Photochem Photobiol B. 2017 May;170:16-24
Zearalenone (ZEN) is a mycotoxin produced mainly by Fusarium species. Fungal contamination of cereals and plants can result in the formation of ZEN, leading to its presence in different foods, animal feeds, and drinks. Because ZEN is an endocrine disruptor, it causes reproductive disorders in farm animals and hyperoestrogenic syndromes in humans. Despite toxicokinetic properties of ZEN were studied in more species, we have no information regarding the interaction of ZEN with serum albumin. Since albumin commonly plays an important role in the toxicokinetics of different toxins, interaction of ZEN with albumin has of high biological importance. Therefore the interaction of ZEN with human serum albumin (HSA) was investigated using spectroscopic methods, ultrafiltration, and molecular modeling studies. Fluorescence spectroscopic studies demonstrate that ZEN forms complex with HSA. Binding constant (K) of ZEN-HSA complex was quantified with fluorescence quenching technique. The determined binding constant (logK=5.1) reflects the strong interaction of ZEN with albumin suggesting the potential biological importance of ZEN-HSA complex formation. Based on the results of the investigations with site markers as well as docking studies, ZEN occupies a non-conventional binding site on HSA. Considering the above listed observations, we should keep in mind this interaction if we would like to precisely understand the toxicokinetic behavior of ZEN.
MobiDB-lite: fast and highly specific consensus prediction of intrinsic disorder in proteins
Bioinformatics. 2017 May 1;33(9):1402-1404
Intrinsic disorder (ID) is established as an important feature of protein sequences. Its use in proteome annotation is however hampered by the availability of many methods with similar performance at the single residue level, which have mostly not been optimized to predict long ID regions of size comparable to domains.
Here, we have focused on providing a single consensus-based prediction, MobiDB-lite, optimized for highly specific (i.e. few false positive) predictions of long disorder. The method uses eight different predictors to derive a consensus which is then filtered for spurious short predictions. Consensus prediction is shown to outperform the single methods when annotating long ID regions. MobiDB-lite can be useful in large-scale annotation scenarios and has indeed already been integrated in the MobiDB, DisProt and InterPro databases.
Availability and Implementation:
MobiDB-lite is available as part of the MobiDB database from URL: http://mobidb.bio.unipd.it/. An executable can be downloaded from URL: http://protein.bio.unipd.it/mobidblite/.
Supplementary data are available at Bioinformatics online.
Unrevealed part of myosin's powerstroke accounts for high efficiency of muscle contraction
Biochim Biophys Acta. 2017 Sep;1861(9):2325-2333
Myosin II, the motor protein driving muscle contraction, uses energy of ATP hydrolysis to produce movement along actin. The key step of energy transduction is the powerstroke, involving rotation of myosin's lever while myosin is attached to actin. Macroscopic measurements indicated high thermodynamic efficiency for energy conversion. However, single-molecule experiments indicated lower efficiency, provoking a long-standing discrepancy.
Based on the Fluctuation-Dissipation Theorem, we built a sufficiently detailed but low degree-of-freedom model reconstructing the entire mechanoenzymatic cycle.
We show that a high axial stiffness of the lever during an initial, experimentally yet unrevealed part of the powerstroke results in a short-time, ratchet-like Kramers effect, and is responsible for the missing efficiency. The second part of the powerstroke is an Eyring-like relaxation that dominantly contributes to lever rotation, but produces only a minor part of the work.
The model reveals the structural background of myosin's capability to function as a robust molecular engine and a very precise load sensor as well. Our model also suggests an explanation for the malfunction of myosins harboring mutations that lead to hypertrophic cardiomyopathies with most severe clinical prognosis.
The model explains how a force-transmitting device within a biological motor can enable high energetic efficiency.
Regulation of the Equilibrium between Closed and Open Conformations of Annexin A2 by N-Terminal Phosphorylation and S100A4-Binding
Structure. 2017 Aug 1;25(8):1195-1207.e5.
Annexin A2 (ANXA2) has a versatile role in membrane-associated functions including membrane aggregation, endo- and exocytosis, and it is regulated by post-translational modifications and protein-protein interactions through the unstructured N-terminal domain (NTD). Our sequence analysis revealed a short motif responsible for clamping the NTD to the C-terminal core domain (CTD). Structural studies indicated that the flexibility of the NTD and CTD are interrelated and oppositely regulated by Tyr24 phosphorylation and Ser26Glu phosphomimicking mutation. The crystal structure of the ANXA2-S100A4 complex showed that asymmetric binding of S100A4 induces dislocation of the NTD from the CTD and, similar to the Ser26Glu mutation, unmasks the concave side of ANXA2. In contrast, pTyr24 anchors the NTD to the CTD and hampers the membrane-bridging function. This inhibition can be restored by S100A4 and S100A10 binding. Based on our results we provide a structural model for regulation of ANXA2-mediated membrane aggregation by NTD phosphorylation and S100 binding.
Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein
FEBS J. 2017 284(21):3637-3661
Human ileal bile acid-binding protein (I-BABP) has a key role in the intracellular transport and metabolic targeting of bile salts. Similar to other members of the family of intracellular lipid-binding proteins (iLBPs), disorder-order transitions and local unfolding processes are thought to mediate ligand entry and release in human I-BABP. To gain insight into the stability of various protein regions, the temperature response of human I-BABP was investigated using NMR, CD and fluorescence spectroscopy, as well as molecular dynamics (MD) simulations. A joint analysis of NMR thermal melting and relaxation dispersion data indicates a complex pattern of internal dynamics with a dominating single barrier and a likely presence of rapidly exchanging conformational substates on both sides of the barrier. Moreover, our residue-specific analysis uncovers a partially unfolded U* state in which part of the helical region with three proximate β-strands contains a substantial amount of residual structure, whereas several segments of the C-terminal half exhibit a high susceptibility to temperature elevation. Cluster analysis of atomic temperature responses indicates a thermodynamic coupling between distant protein sites including the bottom of the β-barrel, the E-F region and part of the helical cap. MD simulations up to 1 μs show correlated motions in the same protein regions and together with the NMR data suggest a role for the highly dynamic D-E turn and E-F region in the initiation of unfolding. The response of human I-BABP to temperature elevation is discussed in the context of the folding/unfolding behaviour of different members of the iLBP family.
A comprehensive assessment of long intrinsic protein disorder from the DisProt database
Bioinformatics. 2017 Sep 18. doi: 10.1093/bioinformatics/btx590
Intrinsic disorder (ID), i.e. the lack of a unique folded conformation at physiological conditions, is a common feature for many proteins, which requires specialized biochemical experiments that are not high-throughput. Missing X-ray residues from the PDB have been widely used as a proxy for ID when developing computational methods. This may lead to a systematic bias, where predictors deviate from biologically relevant ID. Large benchmarking sets on experimentally validated ID are scarce. Recently, the DisProt database has been renewed and expanded to include manually curated ID annotations for several hundred new proteins. This provides a large benchmark set which has not yet been used for training ID predictors.
Here, we describe the first systematic benchmarking of ID predictors on the new DisProt dataset. In contrast to previous assessments based on missing X-ray data, this dataset contains mostly long ID regions and a significant amount of fully ID proteins. The benchmarking shows that ID predictors work quite well on the new dataset, especially for long ID segments. However, a large fraction of ID still goes virtually undetected and the ranking of methods is different than for PDB data. In particular, many predictors appear to confound ID and regions outside x-ray structures. This suggests that the ID prediction methods capture different flavors of disorder and can benefit from highly accurate curated examples.
RecQ helicase triggers a binding mode change in the SSB-DNA complex to efficiently initiate DNA unwinding
Nucleic Acids Res. 2017 Oct 20. doi: 10.1093/nar/gkx939
The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.
Prediction of protein disorder based on IUPred
Protein Sci. 2017 Oct 27. doi: 10.1002/pro.3334
Many proteins contain intrinsically disordered regions (IDRs), functional polypeptide segments that in isolation adopt a highly flexible conformational ensemble instead of a single, well-defined structure. Disorder prediction methods, which can discriminate ordered and disordered regions from the amino acid sequence, have contributed significantly to our current understanding of the distinct properties of IDPs by enabling the characterization of individual examples as well as large-scale analyses of these protein regions. One popular method, IUPred provides a robust prediction of protein disorder based on an energy estimation approach that captures the fundamental difference between the biophysical properties of ordered and disordered regions. This paper reviews the energy estimation method underlying IUPred and the basic properties of the web server. Through an example, it also illustrates how the prediction output can be interpreted in a more complex case by taking into account the heterogeneous nature of IDRs. Various applications that benefited from IUPred to provide improved disorder predictions, complementing domain annotations and aiding the identification of functional short linear motifs are also described here. IUPred is freely available for noncommercial users through the web server (http://iupred.enzim.hu and http://iupred.elte.hu) . The program can also be downloaded and installed locally for large-scale analyses.
Dynamic control of RSK complexes by phosphoswitch-based regulation
FEBS J. 2017 Oct 30. doi: 10.1111/febs.14311.
Assembly and disassembly of protein-protein complexes needs to be dynamically controlled and phosphoswitches based upon linear motifs are crucial in this process. Extracellular signal regulated kinase 2 (ERK2) recognizes a linear binding motif at the C-terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain binding motif which binds RSK substrates (e.g. MAGI-1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2-RSK1 and RSK1-PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based upon linear motifs can be functionally classified as ON and OFF protein-protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear binding motifs. Moreover, interaction of phosphorylated residues with positively-charged residues in disordered regions is likely to be a common mechanism of phosphoregulation.
Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels
Front Physiol. 2017 Jan 9;7:659
Inherited cardiomyopathies are a common form of heart disease that are caused by mutations in sarcomeric proteins with beta cardiac myosin (MYH7) being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. There has been substantial progress in developing assays to measure the force and velocity properties of purified cardiac muscle myosin but it is still challenging to correlate results from molecular and tissue-level experiments. Mutations that cause hypertrophic cardiomyopathy are more common than mutations that lead to dilated cardiomyopathy and are also often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of time myosin spends bound to actin during its ATPase cycle) has been proposed for the treatment of hypertrophic cardiomyopathy. Para-Nitroblebbistatin is a small molecule drug proposed to decrease the duty ratio of class II myosins. We examined the impact of this drug on human beta cardiac myosin using purified myosin motor assays and studies of permeabilized muscle fiber mechanics. We find that with purified human beta-cardiac myosin para-Nitroblebbistatin slows actin-activated ATPase and in vitro motility without altering the ADP release rate constant. In permeabilized human myocardium, para-Nitroblebbistatin reduces isometric force, power, and calcium sensitivity while not changing shortening velocity or the rate of force development (ktr). Therefore, designing a drug that reduces the myosin duty ratio by inhibiting strong attachment to actin while not changing detachment can cause a reduction in force without changing shortening velocity or relaxation.