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.

• Keywords
• 14‐3‐3 proteins, extracellular signal‐regulated kinase 2, microtubule‐associated protein, nuclear magnetic resonance, protein kinase A,
• MeSH
• Phosphorylation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Mitogen-Activated Protein Kinase 1 metabolism   genetics   MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   genetics   MeSH
• 14-3-3 Proteins * metabolism   chemistry   genetics   MeSH
• Microtubule-Associated Proteins * metabolism   chemistry   genetics   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• MAPK1 protein, human MeSH Browser
• Mitogen-Activated Protein Kinase 1 MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins * MeSH
• Microtubule-Associated Proteins * MeSH
• YWHAZ protein, human MeSH Browser

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.

• Keywords
• A-kinase anchoring protein (AKAP), growth factor receptor-bound protein 2 (GRB2), microtubule associated protein (MAP) 2, nuclear magnetic resonance (NMR), protein kinase A (PKA),
• MeSH
• GRB2 Adaptor Protein * metabolism   chemistry   MeSH
• Humans MeSH
• Protein Domains MeSH
• Microtubule-Associated Proteins * metabolism   chemistry   genetics   MeSH
• Proto-Oncogene Proteins c-fyn metabolism   chemistry   genetics   MeSH
• Signal Transduction MeSH
• src Homology Domains MeSH
• Protein Binding * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• GRB2 Adaptor Protein * MeSH
• GRB2 protein, human MeSH Browser
• Microtubule-Associated Proteins * MeSH
• Proto-Oncogene Proteins c-fyn MeSH

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.

• Keywords
• NMR, PKA, Src homology 3 domain, cyclin-dependent kinase, extracellular signal–regulated kinase, growth factor receptor-bound protein 2 (GRB2), microtubule-associated protein,
• MeSH
• Extracellular Signal-Regulated MAP Kinases * metabolism   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Cell Line, Tumor MeSH
• Proline-Directed Protein Kinases * metabolism   MeSH
• Microtubule-Associated Proteins * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK2 protein, human MeSH Browser
• Extracellular Signal-Regulated MAP Kinases * MeSH
• MAP2 protein, human MeSH Browser
• MAPK1 protein, human MeSH Browser
• Proline-Directed Protein Kinases * MeSH
• Microtubule-Associated Proteins * MeSH

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.

• MeSH
• Protein Conformation MeSH
• Reproducibility of Results MeSH
• Molecular Dynamics Simulation MeSH
• Intrinsically Disordered Proteins * MeSH
• Water MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Intrinsically Disordered Proteins * MeSH
• Water MeSH

The stability and dynamics of cytoskeleton in brain nerve cells are regulated by microtubule associated proteins (MAPs), tau and MAP2. Both proteins are intrinsically disordered and involved in multiple molecular interactions important for normal physiology and pathology of chronic neurodegenerative diseases. Nuclear magnetic resonance and cryo-electron microscopy recently revealed propensities of MAPs to form transient local structures and long-range contacts in the free state, and conformations adopted in complexes with microtubules and filamentous actin, as well as in pathological aggregates. In this paper, we compare the longest, 441-residue brain isoform of tau (tau40), and a 467-residue isoform of MAP2, known as MAP2c. For both molecules, we present transient structural motifs revealed by conformational analysis of experimental data obtained for free soluble forms of the proteins. We show that many of the short sequence motifs that exhibit transient structural features are linked to functional properties, manifested by specific interactions. The transient structural motifs can be therefore classified as molecular recognition elements of tau40 and MAP2c. Their interactions are further regulated by post-translational modifications, in particular phosphorylation. The structure-function analysis also explains differences between biological activities of tau40 and MAP2c.

• Keywords
• intrinsically disordered protein, microtubule associated protein, nuclear magnetic resonance, phosphorylation, tau,
• MeSH
• Humans MeSH
• Microtubules chemistry   metabolism   MeSH
• Microtubule-Associated Proteins chemistry   metabolism   MeSH
• tau Proteins chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Microtubule-Associated Proteins MeSH
• tau Proteins 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.

• Keywords
• 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
• X-Ray Diffraction MeSH
• Phosphorylation MeSH
• Protein Conformation * MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• Plectin chemistry   metabolism   MeSH
• Microtubule-Associated Proteins chemistry   metabolism   MeSH
• src Homology Domains MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MAP2 protein, human MeSH Browser
• PLEC protein, human MeSH Browser
• Plectin MeSH
• Microtubule-Associated Proteins 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.

• Keywords
• Intrinsically disordered proteins, Non-uniform sampling, Nuclear magnetic resonance, Relaxation,
• MeSH
• Bacillus subtilis enzymology   MeSH
• DNA-Directed RNA Polymerases chemistry   MeSH
• Nitrogen Isotopes MeSH
• Carbon Isotopes MeSH
• Nuclear Magnetic Resonance, Biomolecular methods   MeSH
• Intrinsically Disordered Proteins chemistry   MeSH
• Hydrogen MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Carbon-13 MeSH Browser
• DNA-Directed RNA Polymerases MeSH
• Nitrogen Isotopes MeSH
• Carbon Isotopes MeSH
• Nitrogen-15 MeSH Browser
• Intrinsically Disordered Proteins MeSH
• Hydrogen 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.

• Keywords
• 14-3-3 protein, mass spectrometry (MS), microtubule-associated protein (MAP), nuclear magnetic resonance (NMR), protein kinase A (PKA),
• MeSH
• Amino Acid Motifs MeSH
• Phosphorylation MeSH
• Mass Spectrometry MeSH
• Kinetics MeSH
• Rats MeSH
• Magnetic Resonance Spectroscopy MeSH
• Microtubules metabolism   MeSH
• Neurons metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• 14-3-3 Proteins chemistry   MeSH
• Microtubule-Associated Proteins chemistry   MeSH
• tau Proteins chemistry   MeSH
• Signal Transduction MeSH
• Tubulin metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• MAP2 protein, rat MeSH Browser
• Mapt protein, rat MeSH Browser
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• Microtubule-Associated Proteins MeSH
• tau Proteins 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.

• Keywords
• ADME, drug delivery systems, biological chemistry, biomaterials, chemical biology, drug design, nanoparticles, natural compounds, proteins and nucleic acids, synthesis, targeting,
• MeSH
• Epigenesis, Genetic MeSH
• Chemistry, Pharmaceutical methods   MeSH
• Drug Delivery Systems MeSH
• Proteins chemistry   MeSH
• Drug Design MeSH
• Systems Biology MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Congress MeSH
• Names of Substances
• Proteins MeSH