Dimerization Affinity Research Articles

Mechanisms of amphibian arrestin 1 self-association and dynamic distribution in retinal photoreceptors

Visual arrestin 1 (Arr1) is an essential protein for termination of the light response in photoreceptors. While mammalian Arr1s form dimers and tetramers at physiological concentrations in vitro, oligomerization in other vertebrates has not been studied. Here we examine self-association of Arr1 from two amphibian species, Xenopus laevis (xArr1), and Ambystoma tigrinum (salArr1). Sedimentation velocity analytical ultracentrifugation showed that xArr1 and salArr1 oligomerization is limited to dimers. The KD for dimer formation was 53 μM for xArr1 and 44 μM for salArr1, similar to the 69 μM KD for bovine Arr1 (bArr1) dimers. Mutations of orthologous amino acids important for mammalian Arr1 oligomerization had no impact on xArr1 dimerization. Crystallography showed that the fold of xArr1 closely resembles that of bArr1 and crystal structures in different space groups revealed two potential xArr1 dimer forms: a symmetrical dimer with a C-domain interface (CC dimer), resembling the bArr1 solution dimer, and an asymmetric dimer with an N-domain/C-domain interface. Mutagenesis of residues predicted to interact in either of these two dimer forms yielded modest reduction in dimer affinity, suggesting that the dimer interfaces compete or are not unique. Indeed, small-angle X-ray scattering and protein painting data were consistent with a symmetric anti-parallel solution dimer (AP dimer) distinct from the assemblies observed by crystallography. Finally, a computational model evaluating xArr1 binding to compartment-specific partners and partitioning based on heterogeneity of available cytoplasmic spaces shows that Arr1 distribution in dark adapted photoreceptors is largely explained by the excluded volume effect together with tuning by oligomerization.

Non-invasive strategy: Developing a topical IL-4Rα-specific nanobody for the treatment of allergic airway diseases

Inhibiting IL-4 and IL-13 are critical cytokines that induce the pathogenic responses of allergic airway diseases. Currently, monoclonal antibodies targeting IL-4Rα are administered subcutaneously to treat eosinophilic rhinosinusitis and allergic asthma. However, these treatments have several drawbacks. To address these issues, we have developed a novel IL-4Rα-targeting nanobody designed for non-invasive delivery to local inflammatory sites in allergic airway diseases. H5, selected via the ribosomal display applied screening from synthetic nanobody library, underwent dimerization and in-silico affinity maturation using AlphaFold2 and GROMACS resulting in a substantial/dramatic enhancement of its binding affinity. H5 effectively controlled inflammatory markers such as MUC5AC, CCL26, and FOXJ1 in human nasal epithelial cells (HNECs) by inhibiting IL-4 and IL-13 signaling. The bivalent form of H5 showed efficacy in easily accessible cells, such as multi-ciliated cells, while the monovalent variant targeted hard-to-reach cells, such as basal cells of HNECs. In summary, we developed a nanobody that could effectively inhibit inflammatory signaling in HNECs via intranasal administration, showing promise as a non-invasive rhinitis treatment.

A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator of Acinetobacter resistance and virulence.

The nosocomial pathogen Acinetobacter baumannii is a major threat to human health. The sensor kinase-response regulator system, BfmS-BfmR, is essential to multidrug resistance and virulence in the bacterium and represents a potential antimicrobial target. Important questions remain about how the system controls resistance and pathogenesis. Although BfmR knockout alters expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls the regulator is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and in vitro phosphorylation to study the functions of phospho-BfmR. We show that phosphorylation is required for BfmR-mediated gene regulation, antibiotic resistance, and sepsis development in vivo. Consistent with activating the protein, phosphorylation induces dimerization and target DNA affinity. Integrated analysis of genome-wide binding and transcriptional profiles of BfmR led to additional key findings: (1) Phosphorylation dramatically expands the number of genomic sites BfmR binds; (2) DNA recognition involves a direct repeat motif widespread across promoters; (3) BfmR directly regulates 303 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (pilus biogenesis); (4) BfmR controls several non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving A. baumannii disease and disentangle the extensive pathogenic gene-regulatory network under its control.

Optimized PAR-2 RING dimerization mediates cooperative and selective membrane binding for robust cell polarity

Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.

NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Abstract LB_B10: Non-V600 BRAF mutants and their precise targeting

Abstract Hyperactive RAS/RAF/MEK/ERK signalling is responsible for a large portion of cancers and targeting this signalling pathway has achieved a promising outcome in clinic cancer treatment. However, significant challenges such as drug resistance are still existing. To resolve these problems, we need a deeper understanding of the regulatory mechanism and hence develop better targeting approaches against this signalling pathway. In this study, we investigated the molecular mechanism that controls that activity of BRAF, a key component of this signalling pathway, and found that BRAF cycled between inactive monomeric status and active dimeric status, which is coordinatively regulated by Cdc37/Hsp90 chaperone and 14-3-3 scaffold. Further, we found that most non-V600 mutations of BRAF in cancer genomes undermined this regulatory mechanism through disturbing the association of BRAF with Cdc37/Hsp90 chaperone or 14-3-3 scaffold and hence trapped BRAF in active dimeric status. Given the high dimer affinity of these non-V600 BRAF mutants, we next examined whether their activity could be inhibited effectively RAF dimer breaker PLX8394 in vitro and in vivo. Indeed, we found that in contrast to clinic RAF inhibitor Vemurafenib (PLX4720), PLX8394 strongly inhibited the activity of non-V600 BRAF mutants and induced a rapid regression of fibroblastomas driven by non-V600 BRAF mutants. Together, our data clearly illustrates how oncogenic non-V600 mutations break down the regulation of BRAF by Cdc37/Hsp90 chaperone and 14-3-3 scaffold, and how these BRAF mutations could be precisely targeted by RAF dimer breaker, which has important implications for anti-BRAF cancer therapy. Citation Format: Jiancheng Hu. Non-V600 BRAF mutants and their precise targeting [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B10.

Theoretical analysis reveals a role for RAF conformational autoinhibition in paradoxical activation.

RAF kinase inhibitors can, under certain conditions, increase RAF kinase signaling. This process, which is commonly referred to as 'paradoxical activation' (PA), is incompletely understood. We use mathematical and computational modeling to investigate PA and derive rigorous analytical expressions that illuminate the underlying mechanism of this complex phenomenon. We find that conformational autoinhibition modulation by a RAF inhibitor could be sufficient to create PA. We find that experimental RAF inhibitor drug dose-response data that characterize PA across different types of RAF inhibitors are best explained by a model that includes RAF inhibitor modulation of three properties: conformational autoinhibition, dimer affinity, and drug binding within the dimer (i.e., negative cooperativity). Overall, this work establishes conformational autoinhibition as a robust mechanism for RAF inhibitor-driven PA based solely on equilibrium dynamics of canonical interactions that comprise RAF signaling and inhibition.

Construction of photoswitchable urea-based multiple H-bonding motifs

In this paper, a series of photoactive urea-based multiple H-bonding motifs were rationally constructed by tethering the photoisomerizable azobenzene units aside to the urea groups of several typical urea-constituted H-bonding arrays. Taking the integrated azo units as photoswitches, their phototriggered E/Z isomerization can be used to reversibly manipulate the locking (with intramolecular H-bonds) and unlocking (without intramolecular H-bonds) of the urea H-bonding sites of these motifs, respectively. This feature enables the E/Z photoisomeric urea derivatives to exhibit switchable intermolecular H-bonding affinities, thus achieving unique photocontrolled self/hetero complementary multiple H-bonded dimerization upon irradiation by different light sources. Benefiting from the wide application of urea group, the development of such urea-based multiple H-bonding system with photoswitchable dimerization affinity is promising in providing a generally applicable approach for the construction of copious urea-based H-bonded supramolecular assembly systems and materials with photoregulable properties and functions.

Single-molecule spectroscopy of the SARS-CoV-2 nucleocapsid protein.

The SARS-CoV-2 nucleocapsid (N) protein plays a variety of roles in the viral life cycle including replication, transcription, and genome packaging. N protein contains two folded domains - the RNA-binding domain and the dimerization domain- and three intrinsically disordered regions located at the N and C terminals and in the linker between the folded domains. Here, we harnessed single-molecule spectroscopy and all atom simulations to investigate the conformational ensemble of the protein and its protein-protein and protein-RNA interactions. Single-molecule Foerster Resonance Energy Transfer (FRET) and nanosecond Fluorescence Correlation Spectroscopy (ns-FCS) experiments support that all three disordered regions are flexible and dynamic in the context of the full-length protein. The estimated conformations are in good agreement with all-atom simulations, which reveal transient helicity regions that may act as local binding interfaces for protein-protein or protein-RNA interactions. We further investigate the conformations of the dimerization domain and its dimerization affinity. We have found that the dimerization domain adopts more expanded configurations in its monomeric form, folds upon dimerization, and has a strong dimerization affinity (Kdim = 30 ± 6 nM). Finally, we have started to investigate how disordered regions contribute to N protein binding of RNA. We focused on the interactions with the RNA-binding domain (RBD) and the N-terminal domain (NTD) tail. Our results indicate that the NTD-RBD region has a preferential interaction with single-stranded RNA, forms a dynamic complex with the nucleic acid, and increases the binding affinity of RBD by about 50-fold. Overall, our data support a model where disordered regions are directly involved in modulating intra- and inter-molecular interactions of the protein.

Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

Computational insights into the differentiated binding affinities of Myc, Max, and Omomyc dimers to the E-boxes of DNA.

Myc is a bHLHZip protein involved in growth control and cancer, which does not form a homodimer. Myc operates in a network with its heterodimerization partner Max, the latter of which can form homodimer and heterodimer. Omomyc, a polypeptide, can block Myc to treat cancers because it can both homodimerize as efficiently as Max and heterodimerize with both Myc and Max. However, the binding efficiencies to DNA for the mentioned two homodimers (Omomyc-Omomyc and Max-Max) and three heterodimers (Myc-Max, Omomyc-Myc, and Omomyc-Max) are still controversial. By molecular dynamics simulations and MM/GBSA free energy calculation, we ranked the binding affinities of five dimers to DNA and analyzed the contribution of single amino acids to the molecular recognition of dimers to DNA. Our simulation showed that the Omomyc-Omomyc dimer exhibited the highest binding energy to DNA, followed by the Omomyc-Myc, Max-Max, Omomyc-Max, and Myc-Max dimers. Moreover, five Arg residues (i.e., 7, 8, 15, 17, and 18 numbered by Omomyc) and five Lys residues (i.e., 6, 22, 40, 43, and 48 numbered by Omomyc) dominated the binding of various dimers to DNA while the residues Asp23 and Asp37 weakened the affinities via repulsive interaction. Our simulation would provide worthy information for further development of the structure-based design of novel Omomyc-like peptide inhibitors against Myc in the future.

Dimerization of cholesterol permits its binding to lipid-free apoA-I

Background and Aims : To model the interaction of lipid-free apoA-I with cholesterol molecules that exist in various self-associated forms in extracellular space. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol.Methods: The interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer was modelled with Schrödinger package. Two crystal structures of 1-43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185-243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were exploited.Results: Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy, still insufficient to overcome the thermodynamic restriction by cholesterol micellization (-52.8 kJ/mol). However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding for both B and M structures exceed the free energy of cholesterol micellization.Conclusions: Cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux. Background and Aims : To model the interaction of lipid-free apoA-I with cholesterol molecules that exist in various self-associated forms in extracellular space. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Methods: The interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer was modelled with Schrödinger package. Two crystal structures of 1-43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185-243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were exploited. Results: Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy, still insufficient to overcome the thermodynamic restriction by cholesterol micellization (-52.8 kJ/mol). However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding for both B and M structures exceed the free energy of cholesterol micellization. Conclusions: Cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.

Optimization of BRET saturation assays for robust and sensitive cytosolic protein–protein interaction studies

Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein–protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (A:D) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending A:D expression detection range and normalizing A:D expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

Exchange Functionals and Basis Sets for Density Functional Theory Studies of Water Splitting on Selected ZnO Nanocluster Catalysts.

In this communication, we use density functional theory (DFT) to study the structural (geometry) and electronic properties (vertical detachment energy and electron affinity) of ZnO monomers and dimers that can be used to form ZnO clusters of different sizes, with a view to adapting one or more of them as catalysts or photocatalysts, standing alone or on suitable substrates like graphene, to split water. We also investigate different pairs of exchange functionals and basis sets to optimize their choice in our DFT calculations and to compare the singlet–triplet energy gaps of small ZnO clusters of different sizes to select an optimal cluster size for water splitting. We find that the B3LYP/DGDZVP2 exchange functional/basis set is a reliable combination for use with DFT to calculate the geometry and electronic properties of small ZnO nanoclusters from among several other combinations of exchange functionals and basis sets. Comparisons of the singlet–triplet energy gaps show that the trimer (ZnO)3 has an energy gap of 58.66 k cal/mol. which is approximately equal to the energy of a visible photon at a wavelength of 500 nm, and a HOMO–LUMO gap of 4.4 eV, making it a suitable choice of photocatalyst for the oxidation of water from among six (ZnO)n nanoclusters of monomers, with n ranging from 1 to 6. We used this exchange functional/basis set to study the structural and energetic details of hydration and hydrolysis of water absorbed on the (ZnO)3 nanocatalyst and calculated the corresponding potential energy profiles to identify three sets of singlet–triplet pathways for water splitting. Detailed study of a pathway showed that oxygen is produced after hydrogen, and the rate-determining step is the formation of hydrogen.

First Principles Calculation of Protein-Protein Dimer Affinities of ALS-Associated SOD1 Mutants.

Cu,Zn superoxide dismutase (SOD1) is a 32 kDa homodimer that converts toxic oxygen radicals in neurons to less harmful species. The dimerization of SOD1 is essential to the stability of the protein. Monomerization increases the likelihood of SOD1 misfolding into conformations associated with aggregation, cellular toxicity, and neuronal death in familial amyotrophic lateral sclerosis (fALS). The ubiquity of disease-associated mutations throughout the primary sequence of SOD1 suggests an important role of physicochemical processes, including monomerization of SOD1, in the pathology of the disease. Herein, we use a first-principles statistical mechanics method to systematically calculate the free energy of dimer binding for SOD1 using molecular dynamics, which involves sequentially computing conformational, orientational, and separation distance contributions to the binding free energy. We consider the effects of two ALS-associated mutations in SOD1 protein on dimer stability, A4V and D101N, as well as the role of metal binding and disulfide bond formation. We find that the penalty for dimer formation arising from the conformational entropy of disordered loops in SOD1 is significantly larger than that for other protein–protein interactions previously considered. In the case of the disulfide-reduced protein, this leads to a bound complex whose formation is energetically disfavored. Somewhat surprisingly, the loop free energy penalty upon dimerization is still significant for the holoprotein, despite the increased structural order induced by the bound metal cations. This resulted in a surprisingly modest increase in dimer binding free energy of only about 1.5 kcal/mol upon metalation of the protein, suggesting that the most significant stabilizing effects of metalation are on folding stability rather than dimer binding stability. The mutant A4V has an unstable dimer due to weakened monomer-monomer interactions, which are manifested in the calculation by a separation free energy surface with a lower barrier. The mutant D101N has a stable dimer partially due to an unusually rigid β-barrel in the free monomer. D101N also exhibits anticooperativity in loop folding upon dimerization. These computational calculations are, to our knowledge, the most quantitatively accurate calculations of dimer binding stability in SOD1 to date.

Quantitation of Human 14-3-3[formula omitted] Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria

14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer–monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.

Structural, spectroscopic (IR, Raman, and NMR), quantum chemical, and molecular docking analysis of (E)-2-(2,5-dimethoxybenzylidene)hydrazinecarbothioamide and its dimers

Thiosemicarbazides are an important class of compounds with pronounced biological activities. In this contribution, the crystallographic structure of (E)-2-(2,5-dimethoxybenzylidene)hydrazinecarbothioamide (DBH) was described, and further spectroscopic studies (IR, Raman, 1H, and 13C NMR) studies were performed. Several density functional theory functionals (B3LYP, CAM-B3LYP, APFD, PBEPBE, M05-2X, and M06-2X) in conjunction with 6-311++G(d,p) were applied for the optimization of the structure. The highest resemblance to the crystallographic structure was obtained for structure optimized at M05-2X/6-311++G(d,p) level of theory. This structure was further used for the prediction of IR, Raman, and NMR spectra. The detailed vibrational and NMR analysis, with the most prominent bands assigned, proved that the experimental and theoretical spectra match well and that the obtained level of theory was suitable for the description of structure. Special emphasis was put on the analysis of dimers of DBH and water/DMSO-DBH structures to examine specific interactions. Natural Bond Orbital (NBO) and Quantum Atoms in Molecules (QTAIM) theories were applied for the quantification of the strength of these interactions. The most active positions were outlined through the calculation of the Fukui functions. A molecular docking study of DBH was performed towards Polo-like Kinase 1 (PLK1) to investigate the potential antitumor activity and the results were compared to volasertib. Specific interactions and binding affinities of monomers and dimers of DBH were discussed.

Conformational dynamics in interleukin 17A and 17F functional complexes is a key determinant of receptor A affinity and specificity

The proinflammatory cytokines IL-17A and IL-17F have been identified as key drivers of a range of human inflammatory diseases, such as psoriasis, which has led to several therapeutic antibodies targeted at IL-17A. The two cytokines have been shown to tightly associate as functional homo and hetero dimers, which induce signalling via the formation of a cell surface signalling complex with a single copy of both IL-17RA and IL-17RC. Striking differences in affinity have been observed for IL-17RA binding to IL-17AA, IL-17AF and IL-17FF, however, the functional significance and molecular basis for this has remained unclear. We have obtained comprehensive backbone NMR assignments for full length IL-17AA (79%), IL-17AF (93%) and IL-17FF (89%), which show that the dimers adopt almost identical backbone topologies in solution to those observed in reported crystal structures. Analysis of the line widths and intensities of assigned backbone amide NMR signals has revealed striking differences in the conformational plasticity and dynamics of IL-17AA compared to both IL-17AF and IL-17FF. Our NMR data indicate that a number of regions of IL-17AA are interconverting between at least two distinct conformations on a relatively slow timescale. Such conformational heterogeneity has previously been shown to play an important role in the formation of many high affinity protein-protein complexes. The locations of the affected IL-17AA residues essentially coincides with the regions of both IL-17A and IL-17F previously shown to undergo significant structural changes on binding to IL-17RA. Substantially less conformational exchange was revealed by the NMR data for IL-17FF and IL-17AF. We propose that the markedly different conformational dynamic properties of the distinct functional IL-17 dimers plays a key role in determining their affinities for IL-17RA, with the more dynamic and plastic nature of IL-17AA contributing to the significantly tighter affinity observed for binding to IL-17RA. In contrast, the dynamic properties are expected to have little influence on the affinity of IL-17 dimers for IL-17RC, which has recently been shown to induce only small structural changes in IL-17FF upon binding.

Interaction of lipid-free apolipoprotein A-I with cholesterol revealed by molecular modeling

We report the modeling of the interaction of differently self-associated lipid-free apoA-I with cholesterol monomer and tail-to-tail (TT) or face-to-face (FF) cholesterol dimer. Cholesterol dimerization is exploited to reconcile the existing experimental data on cholesterol binding to apoA-I with extremely low critical micelle concentration of cholesterol. Two crystal structures of 1–43 N-truncated apolipoprotein Δ(1-43)A-I tetramer (PDB ID: 1AV1, structure B), 185–243 C-truncated apolipoprotein Δ(185-243)A-I dimer (PDB ID: 3R2P, structure M) were analyzed. Cholesterol monomers bind to multiple binding sites in apoA-I monomer, dimer and tetramer with low, moderate and high energy (−10 to −28 kJ/mol with Schrödinger package), still insufficient to overcome the thermodynamic restriction by cholesterol micellization (−52.8 kJ/mol). The binding sites partially coincide with the putative cholesterol-binding motifs. However, apoA-I monomer and dimer existing in structure B, that contain nonoverlapping and non-interacting pairs of binding sites with high affinity for TT and FF cholesterol dimers, can bind in common 14 cholesterol molecules that correspond to existing values. ApoA-I monomer and dimer in structure M can bind in common 6 cholesterol molecules. The values of respective total energy of cholesterol binding up to 64.5 and 67.0 kJ/mol for both B and M structures exceed the free energy of cholesterol micellization. We hypothesize that cholesterol dimers may simultaneously interact with extracellular monomer and dimer of lipid-free apoA-I, that accumulate at acid pH in atheroma. The thermodynamically allowed apolipoprotein-cholesterol interaction outside the macrophage may represent a new mechanism of cholesterol transport by apoA-I from atheroma, in addition to ABCA1-mediated cholesterol efflux.