Structural basis of binding the unique N-terminal domain of microtubule-associated protein 2c to proteins regulating kinases of signaling pathways.

Isoforms of microtubule associated protein 2 (MAP2) differ from their homologue 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 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.

Changes in Endometriosis-Associated Symptoms Following Immunization against SARS-CoV-2: A Cross-Sectional Study.

Background: There are many reports about variations in the menstrual cycle after infection with SARS-CoV-2 or vaccination against it. However, data on SARS-CoV-2 infection or vaccination-related changes in menstruation-associated endometriosis-typical symptoms such as dysmenorrhea, dyspareunia, dyschezia, dysuria, and bloating are rare or missing. Methods: This retrospective study was performed as an online survey among employees and students at the University Hospital Ulm, Germany. Changes regarding the presence of mentioned symptoms and after immunization (vaccination and/or infection) were evaluated with the McNemar Test. Additionally, the risk factors associated with these changes and associations between a subjectively perceived general change in menstruation and changes in the symptoms were evaluated. Results: A total of 1589 respondents were included in the final analysis. Less than 4% of respondents reported the occurrence of new symptoms that they had not experienced before immunization. Overall, there was a significant reduction in the presence of dysmenorrhea, back pain, dyschezia, bloating, and dyspareunia after immunization against coronavirus (p < 0.001). Only 2.3% of all participants reported to have been diagnosed with endometriosis. Factors associated with changes in endometriosis-typical symptoms following immunization were body mass index, age, endometriosis, and thyroid disease. Conclusions: Our results provide unique data about a reduction in the incidence of endometriosis-associated symptoms as dysmenorrhea, dyschezia, and dyspareunia after immunization against COVID-19.

Disturbances of menstrual cycle after immunization against SARS-CoV-2 and their risk factors: Cross-sectional clinical study.

OBJECTIVE: Abnormalities of the menstrual cycle were reported after infection with SARS-CoV-2 and vaccination against it, but the available data are very heterogeneous, do not reflect intermenstrual variations or regional differences, and their risk factors are missing. METHODS: We performed a survey-based study among 6383 employees and students of Ulm University Hospital in Germany between March 1 and 31, 2021. Attributes of menstrual cycles such as cycle length (CL), menses duration (MD), and bleeding volume (BV) were reported as categorical variables before and after immunization against SARS-CoV-2 (first, second, third vaccination or infection). Additionally, the potential risk factors for cycle changes were evaluated and all participants reported the subjective perception of changes, their duration, and time of occurrence. RESULTS: The final analysis included 1726 participants. CL and BV significantly changed after vaccination, but not MD. The subjective perception showed only slight levels of agreement with the objective changes, with the highest Cohen's kappa for CL. The risk factors for the variations in CL were previous cycle irregularities, and risk factors for the changes in BV were age and body mass index. The combination of vaccines (homogenous or heterogeneous) and different types of immunization (infection and vaccination) had no significant effect on cycle irregularities. CONCLUSION: In summary, immunization against SARS-CoV-2 causes changes in the characteristics of the menstrual cycle, which are mostly temporary. The individual risk factors, but not the type of immunization, can affect the mentioned changes.

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1.

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613-694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1613-694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.

Lipid-Dependent Interaction of Human N-BAR Domain Proteins with Sarcolemma Mono- and Bilayers.

The N-BAR domain of the human Bin1 protein is indispensable for T-tubule biogenesis in skeletal muscles. It binds to lipid mono- and bilayers that mimic the sarcolemma membrane composition, and it transforms vesicles into uniform tubules by generating a decorating protein scaffold. We found that Δ(1-33)BAR, lacking the N-terminal amphipathic helix (H0), and H0 alone bind to sarcolemma monolayers, although both proteins are not able to tubulate sarcolemma vesicles. By variation of the lipid composition, we elucidated the role of PI(4,5)P2, cholesterol, and an asymmetric sarcolemma composition for Bin1-N-BAR binding and sarcolemma tubulation. Our results indicate that Bin1-N-BAR binding is low in the absence of PI(4,5)P2 and it is affected by additional changes in the negative headgroup charge and lipid acyl chain composition. However, it is not dependent on the cholesterol content. The results from Langmuir monolayer experiments are complementary to lipid bilayer studies using electron microscopy that provides information on membrane curvature generation.

Synthetic Receptors for the Recognition and Discrimination of Post-Translationally Methylated Lysines.

Post-translational modifications (PTMs) describe the chemical alteration of proteins after their biosynthesis in ribosomes. PTMs play important roles in cell biology, including the regulation of gene expression, cell-cell interactions and the development of different diseases. A prominent class of PTMs is the side-chain methylation of lysine. For the analysis and discrimination of differently methylated lysines, antibodies are widely used, although methylated peptide and protein targets are known to be particularly difficult to differentiate by antibody-based affinity reagents; an additional challenge can be batch-to-batch reproducibility. The application of mass spectrometry techniques for methyllysine discrimination requires a complex sample preparation procedure and is not suited for working in cells. The desire to overcome the above-mentioned challenges has promoted the development of synthetic receptor molecules that recognise and bind methyllysines. Such "artificial antibodies" are of interest for a number of applications, for example, as reagents in biochemical assays, for the isolation and purification of post-translationally methylated proteins and for the tracking of signalling pathways. Moreover, they offer new approaches in diagnostics and therapy. This review delivers an overview of the broad field of methyllysine binding and covers a wide range of synthetic receptors used for the recognition of methylated lysines, including calixarenes, resorcinarenes, pillararenes, disulfide cyclophanes, cucurbiturils and acyclic receptors.

Multifunctional Efficiency: Extending the Concept of Atom Economy to Functional Nanomaterials.

Green chemistry, in particular, the principle of atom economy, has defined new criteria for the efficient and sustainable production of synthetic compounds. In complex nanomaterials, the number of embedded functional entities and the energy expenditure of the assembly process represent additional compound-associated parameters that can be evaluated from an economic viewpoint. In this Perspective, we extend the principle of atom economy to the study and characterization of multifunctionality in nanocarriers, which we define as "multifunctional efficiency". This concept focuses on the design of highly active nanomaterials by maximizing integrated functional building units while minimizing inactive components. Furthermore, synthetic strategies aim to minimize the number of steps and unique reagents required to make multifunctional nanocarriers. The ultimate goal is to synthesize a nanocarrier that is highly specialized but practical and simple to make. Owing to straightforward crystal engineering, metal-organic framework (MOF) nanoparticles are an excellent example to illustrate the idea behind this concept and have the potential to emerge as next-generation drug delivery systems. Here, we highlight examples showing how the combination of the properties of MOFs ( e.g., their organic-inorganic hybrid nature, high surface area, and biodegradability) and induced systematic modifications and functionalizations of the MOF's scaffold itself lead to a nanocarrier with high multifunctional efficiency.

Different supramolecular interactions mediated by Br atoms in the crystal structures of three anisole derivatives.

Three anisole building blocks featuring bis(hydroxymethyl) or bis(bromomethyl) pendants have been analyzed with regard to their molecular structures and packing behaviour. The compounds are ethyl 3,5-bis(hydroxymethyl)-4-methoxybenzoate, C12H16O5, (I), [5-bromo-3-(hydroxymethyl)-2-methoxyphenyl]methanol [or 4-bromo-2,6-bis(hydroxymethyl)anisole], C9H11BrO3, (II), and 5-bromo-1,3-bis(bromomethyl)-2-methoxybenzene [or 4-bromo-2,6-bis(bromomethyl)anisole], C9H9Br3O, (III). A typical supramolecular pattern involved C-H...π interactions generating molecular stacks, while π-π interactions were only observed in the absence of bromine, indicating a striking influence on the distances between adjacent aromatic moieties. When comparing bis(hydroxymethyl) compound (II) with bis(bromomethyl) compound (III), we found that the strong O-H...O hydrogen bonds in a zigzag arrangement in the first are replaced by C-H...Br interactions in the second without a change in the general packing.

Selective recognition of the di/trimethylammonium motif by an artificial carboxycalixarene receptor.

Chemical tools that recognise post-translational modifications have promising applications in biochemistry and in therapy. We report a simple carboxycalixarene that selectively binds molecules containing di/trimethylammonium moieties in isolation, in cell lysates and when incorporated in histone peptides. Our findings reveal the potential of using carboxycalixarene-based receptors to study epigenetic regulation.

Crystal structures of functional building blocks derived from bis(benzo[b]thiophen-2-yl)methane.

The syntheses of three bis(benzo[b]thiophen-2-yl)methane derivatives, namely bis(benzo[b]thiophen-2-yl)methanone, C17H10OS2, (I), 1,1-bis(benzo[b]thiophen-2-yl)-3-(trimethylsilyl)prop-2-yn-1-ol, C22H20OS2Si, (II), and 1,1-bis(benzo[b]thiophen-2-yl)prop-2-yn-1-ol, C19H12OS2, (III), are described and their crystal structures discussed comparatively. The conformation of ketone (I) and the respective analogues are rather similar for most of the compounds compared. This is true for the interplanar angles, the Caryl-Cbridge-Caryl angles and the dihedral angles. The best resemblance is found for a bioisotere of (I), viz. 2,2'-dinaphthyl ketone, (VII). By way of interest, the crystal packings also reveal similarities between (I) and (VII). In (I), the edge-to-face interactions seen between two napthyl residues in (VII) are substituted by S...π contacts between the benzo[b]thiophen-2-yl units in (I). In the structures of the bis(benzo[b]thiophen-2-yl)methanols, i.e. (II) and (III), the interplanar angles are also quite similar compared with analogues and related active pharmaceutical ingredients (APIs) containing the dithiophen-2-ylmethane scaffold, though the dihedral angles show a larger variability and produce unsymmetrical molecules.

Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.

The 30kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle.

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain.

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.

Consequences of proline-to-alanine substitutions for the stability and refolding of onconase.

Peptidyl-prolyl isomerization reactions can make for rate-limiting steps in protein folding due to their high activation energy. Onconase, an unusually stable ribonuclease A homologue from the Northern leopard frog, contains four trans proline residues in its native state. During the refolding from its guanidine hydrochloride unfolded state, which includes the formation of a folding intermediate, the slowest of the three phases has earlier been attributed to a cis-to-trans peptidyl-prolyl isomerization reaction. We thus substituted all four proline residues individually by alanine and investigated the effect of the amino acid substitutions on the folding and stability of the onconase variants. All onconase variants proved to adopt a tertiary structure comparable with that of the wild-type protein. Although the slow phase was not eliminated for any of the variants, the P43A substitution resulted in an increase in the rate constant of the fast folding phase, i.e. a faster formation of the folding intermediate. This variant also exhibits a significant increase in thermodynamic stability. As residue 43 belongs to those residues that are protected from hydrogen exchange with the solvent in the folding intermediate, the increase in the rate constant and stability of the P43A variant emphasizes the importance of the intermediate for the folding of onconase.

Methyl 6-amino-6-oxohexa-noate.

The title compound, C(7)H(13)NO(3), adopts an approximately planar conformation. The torsion angles in the aliphatic chain between the carbonyl group C atoms range from 172.97 (14) to 179.38 (14)° and the r.m.s. deviation of all non-H atoms is 0.059 Å. The crystal packing is dominated by two strong N-H⋯O hydrogen bonds involving the amide groups and forming R(2) (2)(8) rings and C(4) chains. Overall, a two-dimensional network parallel to (100) is formed. A weak inter-molecular C-H⋯O inter-action is also present.

3-Meth-oxy-3-oxopropanaminium chloride.

In the title compound, C(4)H(10)NO(2) (+)·Cl(-), the central ethyl-ene bond of the cation adopts a gauche conformation. The three H atoms of the -NH(3) (+) group are engaged in strong and highly directional inter-molecular N-H⋯Cl hydrogen bonds, which result in a tape-like arrangement along [010] of the respective ion pairs. In addition, weak inter-molecular C-H⋯Cl and C-H⋯O inter-actions are present.

1-Methyl-4-(4-nitro-benzo-yl)pyridinium perchlorate.

In the main mol-ecule of the title compound, C(13)H(11)N(2)O(3) (+)·ClO(4) (-), the two aromatic rings are twisted by 56.19 (3)° relative to each other and the nitro group is not coplanar with the benzene ring [36.43 (4)°]. The crystal packing is dominated by infinite aromatic stacks in the a-axis direction. These are formed by the benzene units of the mol-ecule featuring an alternating arrangement, which explains the two different distances of 3.3860 (4) and 3.4907 (4) Šfor the aromatic units (these are the perpendicular distances of the centroid of one aromatic ring on the mean plane of the other other aromatic ring). Adjacent stacks are connected by π-π stacking between two pyridinium units [3.5949 (4) Å] and weak C-H⋯O inter-actions. The perchlorate anions are accomodated in the lattice voids connected to the cation via weak C-H⋯O contacts between the O atoms of the anion and various aromatic as well as methyl H atoms.

Bridge-substituted calix[4]arenes: syntheses, conformations and application.

The bridge-substituted calix[4]arene carboxylic acid, 5,11,17,23-tetra-tert-butyl-25,26,27,28-tetramethoxy-calix[4]arene-2-carboxylic acid (1), can be readily converted to various esters 2-4 and reduced to the alcohol 5, which reacts with methyl iodide to give the ether 6. The alcohol can be dansylated to give 7, the fluorescence of which is selectively quenched by Cu(II) in acetonitrile. An attempt to convert the acid 1 to an amide resulted unexpectedly in the formation of a lactone 8. The conformational characteristics of 1-8 have been studied in solution and, in the cases of 2 and 4, in the solid state by determination of their single-crystal X-ray structures. With the exception of 8, in all these compounds the bridge substituent adopts an equatorial (lateral) orientation.

25-Allyloxy-5,11,17,23-tetra-tert-butyl-26,27,28-trihydroxycalix[4]arene chloroform disolvate.

In the title solvated calixarene, C(47)H(60)O(4) x 2 CHCl(3), the host chalice displays an almost undistorted cone conformation, stabilized by three strong O-H...O hydrogen bonds at the calixarene's lower rim. One chloroform solvent molecule is fixed in the calixarene cavity by C-H...pi interactions, while the second is accommodated in a clathrate-like mode in elliptical packing voids. These voids are spanned by six host molecules connected via C-H...pi contacts and van der Waals interactions. Within the crystal structure, one tert-butyl group of the calixarene host is disordered over two orientations, with occupancies of 0.884 (4) and 0.116 (4). Furthermore, both solvent molecules show disorder, with occupancies of 0.857 (2) and 0.143 (2) for the cavitate-type, and 0.9359 (17) and 0.0641 (17) for the clathrate-type chloroform solvent molecules.

1-(Hydroxy-meth-yl)pyrene.

The asymmetric unit of the title compound, C(17)H(12)O, contains two molecules, in which the fused aromatic ring systems are almost planar [maximum deviations = 0.0529 (9) and 0.0256 (9) Å]. In the crystal, aromatic π-π stacking inter-actions (perpendicular distance of centroids of about 3.4 Å) and strong O-H⋯O hydrogen bonds result in a helical arrangement of pyrenyl dimers.

Calix[4]arenes featuring a direct lower rim attachment of dansyl groups. Synthesis, fluorescence properties and first report on crystal structures.

A series of new fluorescent calix[4]arenes (2-7; 5 has been isolated as two not yet described conformational isomers) featuring two directly lower rim attached dansyl moieties besides other lower or upper rim site substituents have been synthesized and investigated with reference to their fluorescence properties including potential sensing capability for metal ions. Except for the nitro substituted compound 7, which showed a moderate fluorescence quenching on the addition of Hg2+ in acetonitrile, the calixarenes 2-6 were found to selectively recognize Cu2+ in the 10(-6) mol L(-1) concentration range detectable by the nearly total quenching of their intrinsic fluorescence. Using fluorescence titration experiments, the ideal complexation ratio [metal ion]/[ligand] for three exemplarily investigated calixarenes (3, 5a and 7) was determined to be 2:1. For 5a, a new red shifted signal was observed by the complexation of Cu2+ while the addition of Hg2+ only yields a moderate quenching of the parent signal, indicating a different binding mode for both metal ions. This finding enables the calixarene 5a to be used as a suitable chemosensor for the simultaneous determination of copper and mercury. In this paper we also present the first crystal structures of dansylated calixarenes having the dansyl groups directly attached to the lower rim site. They involve the unsolvated calixarene 5b and the two solvent inclusion compounds 2.CH2(OH)CN and 3.3CH3CN, each showing an extensive pattern of non-covalent interactions. In both solvates, the calixarenes are fixed in a cone conformation while the unsolvated calixarene 5b adopts the partial cone conformation. A solution 1H NMR study in CDCl3 reveals also the cone conformation for all dansylated calixarenes, except the more conformationally flexible upper rim unsubstituted calixarene 5 which showed cone and partial cone conformers 5a and 5b, respectively, in an approximate 2:3 ratio.