Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

• Klíčová slova
• 14‐3‐3 proteins, extracellular signal‐regulated kinase 2, microtubule‐associated protein, nuclear magnetic resonance, protein kinase A,
• MeSH
• fosforylace MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   genetika   MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   genetika   MeSH
• proteiny 14-3-3 * metabolismus   chemie   genetika   MeSH
• proteiny asociované s mikrotubuly * metabolismus   chemie   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• MAPK1 protein, human MeSH Prohlížeč
• mitogenem aktivovaná proteinkinasa 1 MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 * MeSH
• proteiny asociované s mikrotubuly * MeSH
• YWHAZ protein, human MeSH Prohlížeč

Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.

• Klíčová slova
• NMR, PKA, Src homology 3 domain, cyclin-dependent kinase, extracellular signal–regulated kinase, growth factor receptor-bound protein 2 (GRB2), microtubule-associated protein,
• MeSH
• extracelulárním signálem regulované MAP kinasy * metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteinkinasy řízené prolinem * metabolismus   MeSH
• proteiny asociované s mikrotubuly * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK2 protein, human MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy * MeSH
• MAP2 protein, human MeSH Prohlížeč
• MAPK1 protein, human MeSH Prohlížeč
• proteinkinasy řízené prolinem * MeSH
• proteiny asociované s mikrotubuly * MeSH

Microtubule-associated protein 2c (MAP2c) is a 49-kDa intrinsically disordered protein regulating the dynamics of microtubules in developing neurons. MAP2c differs from its sequence homologue Tau in the pattern and kinetics of phosphorylation by cAMP-dependent protein kinase (PKA). Moreover, the mechanisms through which MAP2c interacts with its binding partners and the conformational changes and dynamics associated with these interactions remain unclear. Here, we used NMR relaxation and paramagnetic relaxation enhancement techniques to determine the dynamics and long-range interactions within MAP2c. The relaxation rates revealed large differences in flexibility of individual regions of MAP2c, with the lowest flexibility observed in the known and proposed binding sites. Quantitative conformational analyses of chemical shifts, small-angle X-ray scattering (SAXS), and paramagnetic relaxation enhancement measurements disclosed that MAP2c regions interacting with important protein partners, including Fyn tyrosine kinase, plectin, and PKA, adopt specific conformations. High populations of polyproline II and α-helices were found in Fyn- and plectin-binding sites of MAP2c, respectively. The region binding the regulatory subunit of PKA consists of two helical motifs bridged by a more extended conformation. Of note, although MAP2c and Tau did not differ substantially in their conformations in regions of high sequence identity, we found that they differ significantly in long-range interactions, dynamics, and local conformation motifs in their N-terminal domains. These results highlight that the N-terminal regions of MAP2c provide important specificity to its regulatory roles and indicate a close relationship between MAP2c's biological functions and conformational behavior.

• Klíčová slova
• NMR relaxation, Tau protein (Tau), microtubule-associated protein (MAP), nuclear magnetic resonance (NMR), paramagnetic relaxation enhancement (PRE), protein conformation, small-angle X-ray scattering (SAXS),
• MeSH
• difrakce rentgenového záření MeSH
• fosforylace MeSH
• konformace proteinů * MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• plektin chemie   metabolismus   MeSH
• proteiny asociované s mikrotubuly chemie   metabolismus   MeSH
• src homologní domény MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAP2 protein, human MeSH Prohlížeč
• PLEC protein, human MeSH Prohlížeč
• plektin MeSH
• proteiny asociované s mikrotubuly MeSH

Description of protein dynamics is known to be essential in understanding their function. Studies based on a well established [Formula: see text] NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of [Formula: see text] chemical shifts very often observed within intrinsically disordered proteins complicates utilization of standard 2D HN correlated spectra because a limited number of amino acids can be characterized. Here we present a suite of triple resonance HNCO-type NMR experiments for measurements of five [Formula: see text] relaxation parameters ([Formula: see text], [Formula: see text], NOE, cross-correlated relaxation rates [Formula: see text] and [Formula: see text]) in doubly [Formula: see text],[Formula: see text]-labeled proteins. We show that the third spectral dimension combined with non-uniform sampling provides relaxation rates for almost all residues of a protein with extremely poor chemical shift dispersion, the C terminal domain of [Formula: see text]-subunit of RNA polymerase from Bacillus subtilis. Comparison with data obtained using a sample labeled by [Formula: see text] only showed that the presence of [Formula: see text] has a negligible effect on [Formula: see text], [Formula: see text], and on the cross-relaxation rate (calculated from NOE and [Formula: see text]), and that these relaxation rates can be used to calculate accurate spectral density values. Partially [Formula: see text]-labeled sample was used to test if the observed increase of [Formula: see text] [Formula: see text] in the presence of [Formula: see text] corresponds to the [Formula: see text] dipole-dipole interactions in the [Formula: see text],[Formula: see text]-labeled sample.

• Klíčová slova
• Intrinsically disordered proteins, Non-uniform sampling, Nuclear magnetic resonance, Relaxation,
• MeSH
• Bacillus subtilis enzymologie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• vodík MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Carbon-13 MeSH Prohlížeč
• DNA řízené RNA-polymerasy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• Nitrogen-15 MeSH Prohlížeč
• vnitřně neuspořádané proteiny MeSH
• vodík MeSH

Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration. Using NMR methods providing single-residue resolution and quantitative comparison, we investigated molecular interactions important for the regulatory roles of MAP2c in microtubule dynamics. We found that MAP2c and Tau significantly differ in the position and kinetics of sites that are phosphorylated by cAMP-dependent protein kinase (PKA), even in highly homologous regions. We determined the binding sites of unphosphorylated and phosphorylated MAP2c responsible for interactions with the regulatory protein 14-3-3ζ. Differences in phosphorylation and in charge distribution between MAP2c and Tau suggested that both MAP2c and Tau respond to the same signal (phosphorylation by PKA) but have different downstream effects, indicating a signaling branch point for controlling microtubule stability. Although the interactions of phosphorylated Tau with 14-3-3ζ are supposed to be a major factor in microtubule destabilization, the binding of 14-3-3ζ to MAP2c enhanced by PKA-mediated phosphorylation is likely to influence microtubule-MAP2c binding much less, in agreement with the results of our tubulin co-sedimentation measurements. The specific location of the major MAP2c phosphorylation site in a region homologous to the muscarinic receptor-binding site of Tau suggests that MAP2c also may regulate processes other than microtubule dynamics.

• Klíčová slova
• 14-3-3 protein, mass spectrometry (MS), microtubule-associated protein (MAP), nuclear magnetic resonance (NMR), protein kinase A (PKA),
• MeSH
• aminokyselinové motivy MeSH
• fosforylace MeSH
• hmotnostní spektrometrie MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• magnetická rezonanční spektroskopie MeSH
• mikrotubuly metabolismus   MeSH
• neurony metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny 14-3-3 chemie   MeSH
• proteiny asociované s mikrotubuly chemie   MeSH
• proteiny tau chemie   MeSH
• signální transdukce MeSH
• tubulin metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• MAP2 protein, rat MeSH Prohlížeč
• Mapt protein, rat MeSH Prohlížeč
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny tau MeSH
• tubulin MeSH

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on August 28-September 1, 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

• Klíčová slova
• ADME, drug delivery systems, biological chemistry, biomaterials, chemical biology, drug design, nanoparticles, natural compounds, proteins and nucleic acids, synthesis, targeting,
• MeSH
• epigeneze genetická MeSH
• farmaceutická chemie metody   MeSH
• lékové transportní systémy MeSH
• proteiny chemie   MeSH
• racionální návrh léčiv MeSH
• systémová biologie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• kongresy MeSH
• Názvy látek
• proteiny MeSH

Protein structures are valuable tools to understand protein function. Nonetheless, proteins are often considered as rigid macromolecules while their structures exhibit specific flexibility, which is essential to complete their functions. Analyses of protein structures and dynamics are often performed with a simplified three-state description, i.e., the classical secondary structures. More precise and complete description of protein backbone conformation can be obtained using libraries of small protein fragments that are able to approximate every part of protein structures. These libraries, called structural alphabets (SAs), have been widely used in structure analysis field, from definition of ligand binding sites to superimposition of protein structures. SAs are also well suited to analyze the dynamics of protein structures. Here, we review innovative approaches that investigate protein flexibility based on SAs description. Coupled to various sources of experimental data (e.g., B-factor) and computational methodology (e.g., Molecular Dynamic simulation), SAs turn out to be powerful tools to analyze protein dynamics, e.g., to examine allosteric mechanisms in large set of structures in complexes, to identify order/disorder transition. SAs were also shown to be quite efficient to predict protein flexibility from amino-acid sequence. Finally, in this review, we exemplify the interest of SAs for studying flexibility with different cases of proteins implicated in pathologies and diseases.

• Klíčová slova
• allostery, disorder, protein complexes, protein folding, protein structures, protein—DNA interactions, secondary structure, structural alphabet,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Microtubule-associated proteins (MAPs) are abundantly present in axons and dendrites, and have been shown to play crucial role during the neuronal morphogenesis. The period of main dendritic outgrowth and synaptogenesis coincides with high expression levels of one of MAPs, the MAP2c, in rats. The MAP2c is a 49.2 kDa intrinsically disordered protein. To achieve an atomic resolution characterization of such a large protein, we have developed a protocol based on the acquisition of two five-dimensional (13)C-directly detected NMR experiments. Our previously published 5D CACONCACO experiment (Nováček et al. in J Biomol NMR 50(1):1-11, 2011) provides the sequential assignment of the backbone resonances, which is not interrupted by the presence of the proline residues in the amino acid sequence. A novel 5D HC(CC-TOCSY)CACON experiment facilitates the assignment of the aliphatic side chain resonances. To streamline the data analysis, we have developed a semi-automated procedure for signal assignments. The obtained data provides the first atomic resolution insight into the conformational state of MAP2c and constitutes a model for further functional studies of MAPs.

• MeSH
• algoritmy MeSH
• glycin MeSH
• krysa rodu Rattus MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny asociované s mikrotubuly chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• vnitřně neuspořádané proteiny chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glycin MeSH
• proteiny asociované s mikrotubuly MeSH
• vnitřně neuspořádané proteiny MeSH

A pair of 4D NMR experiments for the backbone assignment of disordered proteins is presented. The experiments exploit (13)C direct detection and non-uniform sampling of the indirectly detected dimensions, and provide correlations of the aliphatic proton (H(α), and H(β)) and carbon (C(α), C(β)) resonance frequencies to the protein backbone. Thus, all the chemical shifts regularly used to map the transient secondary structure motifs in the intrinsically disordered proteins (H(α), C(α), C(β), C', and N) can be extracted from each spectrum. Compared to the commonly used assignment strategy based on matching the C(α) and C(β) chemical shifts, inclusion of the H(α) and H(β) provides up to three extra resonance frequencies that decrease the chance of ambiguous assignment. The experiments were successfully applied to the original assignment of a 12.8 kDa intrinsically disordered protein having a high content of proline residues (26 %) in the sequence.

• MeSH
• izotopy uhlíku chemie   MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• prolin chemie   MeSH
• proteiny chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izotopy uhlíku MeSH
• prolin MeSH
• proteiny MeSH

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of (13)C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriální proteiny chemie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• izotopy uhlíku MeSH
• konformace proteinů MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• proteiny chemie   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• izotopy uhlíku MeSH
• proteiny MeSH