Binding Specificity of ASHH2 CW Domain Toward H3K4me1 Ligand Is Coupled to Its Structural Stability Through Its α1-Helix

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site—the two tryptophans and the α1-helix form and maintain the binding pocket— were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.publishedVersio

Conformational selection mechanism of ASHH2 methyltransferase CW domain recognising H3K4me1 histone modification

From simple double-stranded molecule DNA is packed in the nucleus via different intermediate structures with help of various proteins organizing a complex structure referred to as chromatin. A subunit of chromatin is a nucleosome that reminiscent a reel in which the DNA region is winded around a core of proteins which are called histones. Special feature of these histones is that their terminal tails are unstructured and protrude out of the nucleosomes. This makes them to be prone to different chemical modifications such as methylation, acetylation, phosphorylation and so on. The modifications have certain effects on the associated genomic regions, for example acetylation leads to activation of genes and methylation is a mark of silencing. The pattern of the modifications and their effects are conserved and shared among organisms and is referred as “histone code”. Regulation of genes at the level of these modifications is named epigenetic regulation. In order to maintain the histone code and allow epigenetic regulation specialized machinery is necessary. Such function is performed by various proteins that are able to recognize and modify these histone modifications. Worth mentioning that if something goes wrong with these processes it leads to development of various diseases and cancer is one of them. The research presented in the dissertation was focusing on the mechanism that allows specific recognition of monomethylated modification of lysine at 4th position on histone H3 (H3K4me1) by CW domain of histone methyltransferase ASHH2. After structural and biophysical characterization of interaction it can be concluded that CW exists as an ensemble of fluctuating structures that sample different modifications of H3K4 until it uptakes H3K4me1 modification that pushes the conformation of the domain in such state that it has the most stable complex which is balanced by formation of additional secondary structure features and has lowest free energy

Conformational selection mechanism of ASHH2 methyltransferase CW domain recognising H3K4me1 histone modification

From simple double-stranded molecule DNA is packed in the nucleus via different intermediate structures with help of various proteins organizing a complex structure referred to as chromatin. A subunit of chromatin is a nucleosome that reminiscent a reel in which the DNA region is winded around a core of proteins which are called histones. Special feature of these histones is that their terminal tails are unstructured and protrude out of the nucleosomes. This makes them to be prone to different chemical modifications such as methylation, acetylation, phosphorylation and so on. The modifications have certain effects on the associated genomic regions, for example acetylation leads to activation of genes and methylation is a mark of silencing. The pattern of the modifications and their effects are conserved and shared among organisms and is referred as “histone code”. Regulation of genes at the level of these modifications is named epigenetic regulation. In order to maintain the histone code and allow epigenetic regulation specialized machinery is necessary. Such function is performed by various proteins that are able to recognize and modify these histone modifications. Worth mentioning that if something goes wrong with these processes it leads to development of various diseases and cancer is one of them. The research presented in the dissertation was focusing on the mechanism that allows specific recognition of monomethylated modification of lysine at 4th position on histone H3 (H3K4me1) by CW domain of histone methyltransferase ASHH2. After structural and biophysical characterization of interaction it can be concluded that CW exists as an ensemble of fluctuating structures that sample different modifications of H3K4 until it uptakes H3K4me1 modification that pushes the conformation of the domain in such state that it has the most stable complex which is balanced by formation of additional secondary structure features and has lowest free energy

Goldilocks Dilemma: LPS Works Both as the Initial Target and a Barrier for the Antimicrobial Action of Cationic AMPs on E. coli

Antimicrobial peptides (AMPs) are generally membrane-active compounds that physically disrupt bacterial membranes. Despite extensive research, the precise mode of action of AMPs is still a topic of great debate. This work demonstrates that the initial interaction between the Gram-negative E. coli and AMPs is driven by lipopolysaccharides (LPS) that act as kinetic barriers for the binding of AMPs to the bacterial membrane. A combination of SPR and NMR experiments provide evidence suggesting that cationic AMPs first bind to the negatively charged LPS before reaching a binding place in the lipid bilayer. In the event that the initial LPS-binding is too strong (corresponding to a low dissociation rate), the cationic AMPs cannot effectively get from the LPS to the membrane, and their antimicrobial potency will thus be diminished. On the other hand, the AMPs must also be able to effectively interact with the membrane to exert its activity. The ability of the studied cyclic hexapeptides to bind LPS and to translocate into a lipid membrane is related to the nature of the cationic charge (arginine vs. lysine) and to the distribution of hydrophobicity along the molecule (alternating vs. clumped tryptophan)

Binding Specificity of ASHH2 CW Domain Toward H3K4me1 Ligand Is Coupled to Its Structural Stability Through Its α1-Helix

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site—the two tryptophans and the α1-helix form and maintain the binding pocket— were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues