Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX–DNMT3–DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino‐terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1α to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.     

H3K36me2/3 Binding and DNA Binding of the DNA Methyltransferase DNMT3A PWWP Domain Both Contribute to its Chromatin Interaction

The PWWP domain of DNMT3 DNA methyltransferases binds to histone H3 tails containing methylated K36, and this activity is important for heterochromatic targeting. Here, we show that the PWWP domain of mouse DNMT3A binds to H3K36me2 and H3K36me3 with a slight preference for H3K36me2. PWWP domains have also been reported to bind to DNA, and the close proximity of H3K36 and nucleosomal DNA suggests a combined binding to H3K36me2/3 and DNA. We show here that the DNMT3A PWWP domain binds to DNA with a weak preference for AT-rich sequences and that the designed charge reversal R362E mutation disrupts DNA binding. The K295E mutation, as well as K295I recently identified in paraganglioma, a rare neuroendocrine neoplasm, disrupts both DNA and H3K36me2/3 binding, which is in agreement with the proximity of K295 to residues involved in K36me2/3 methyllysine binding. Nucleosome pulldown experiments show that DNA binding and H3K36me2/3 binding are important for the interaction of the DNMT3A PWWP domain with nucleosomes. Localization studies of transiently transfected fluorescently-tagged wild-type and PWWP-mutated full-length DNMT3A indicate that both interactions contribute to the subnuclear localization of DNMT3A in mouse cells. In summary, our data demonstrate that the combined binding of the DNMT3A PWWP domain to the H3 tail containing K36me2/3 and to the nucleosomal or linker DNA is important for its chromatin interaction and subnuclear targeting of DNMT3A in living cells.     

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.     

H3K9me2/3 binding of the MBT domain protein LIN-61 is essential for Caenorhabditis elegans vulva development

MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3 binding proteins, LIN-61 and HPL-2, as well as the H3K9MT MET-2 in distinct developmental pathways.     

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.     

Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3.

Of the several proteins that bind along the cytoplasmic domain of erythrocyte membrane band 3, only the sites of interaction of proteins 4.1 and 4.2 remain to be at least partially localized. Using five independent techniques, we have undertaken to map and characterize the binding site of band 4.1 on band 3. First, transfer of a radioactive cross-linker (125I-2-(p-azido-salicylamido)ethyl-1-3-dithiopropionate) from purified band 4.1 to its binding sites on stripped inside-out erythrocyte membrane vesicles (stripped IOVs) revealed major labeling of band 3, glycophorin C, and glycophorin A. Proteolytic mapping of the stripped IOVs then demonstrated that the label on band 3 was confined largely to a fragment comprising residues 1-201. Second, competitive binding experiments with Fab fragments of monoclonal and peptide-specific polyclonal antibodies to numerous epitopes along the cytoplasmic domain of band 3 displayed stoichiometric competition only with Fabs to epitopes between residues 1 and 91 of band 3. Weak competition was also observed with Fabs to a sequence of the cytoplasmic domain directly adjacent to the membrane-spanning domain, but only at 50-100-fold excess of Fab. Third, band 4.1 protected band 3 from chymotryptic hydrolysis at tyrosine 46 and to a much lesser extent at a site within the junctional peptide connecting the membrane-spanning and cytoplasmic domains of band 3. Fourth, ankyrin, which has been previously shown to interact with band 3 both near a putative central hinge and at the N terminus competed with band 4.1 for band 3 in stripped IOVs. Since band 4.1 does not associate with band 3 near the flexible central hinge, the competition with ankyrin can be assumed to derive from a mutual association with the N terminus. Finally, a synthetic peptide corresponding to residues 1-15 of band 3 was found to mildly inhibit band 4.1 binding to stripped IOVs. Taken together, these data suggest that band 4.1 binds band 3 predominantly near the N terminus, with a possible secondary site near the junction of the cytoplasmic domain and the membrane.     

Solution structure of the second SH3 domain of human CMS and a newly identified binding site at the C-terminus of c-Cbl

CMS, cas ligand with multiple Src homology 3 (SH3) domains, belongs to a family of ubiquitously expressed adaptor proteins. Among the CMS binding proteins, c-Cbl has been mostly extensively studied. It was reported that the motif PKPFPR (residues 824-829) of c-Cbl can bind to the N-terminus SH3 domains of CMS. Here we report the solution structure of the second SH3 domain of CMS (CMS_SH3_B), furthermore, we have identified that a peptide from residues 701 to 714 of c-Cbl (Cbl-p), i.e. MTPSSRPLRPLDTS, can specially bind to CMS_SH3_B using NMR chemical shift perturbation, suggesting that the peptide is a new potential CMS binding site. Among the peptide, TPSSRPLR is the core binding motif and Arg709 plays a key role in the interaction. Cbl-p binding interface on CMS_SH3_B along a hydrophobic channel is composed of RT loop, n-Src loop and beta4 strand and divided into three pockets. This work indicates the solution structure of CMS_SH3_B bears the canonical beta-beta-beta-beta-alpha-beta fold and a new binding site in c-Cbl involved in its interaction with CMS, which probably contributes to the clustering of CMS. All the information provided here should be beneficial for the future functional study of CMS.     

Molecular basis of the interaction of Saccharomyces cerevisiae Eaf3 chromo domain with methylated H3K36

Eaf3 is a component of both NuA4 histone acetyltransferase and Rpd3S histone deacetylase complexes in Saccharomyces cerevisiae. It is involved in the regulation of the global pattern of histone acetylation that distinguishes promoters from coding regions. Eaf3 contains a chromo domain at the N terminus that can bind to methylated Lys-36 of histone H3 (H3K36). We report here the crystal structures of the Eaf3 chromo domain in two truncation forms. Unlike the typical HP1 and Polycomb chromo domains, which contain a large groove to bind the modified histone tail, the Eaf3 chromo domain assumes an autoinhibited chromo barrel domain similar to the human MRG15 chromo domain. Compared with other chromo domains, the Eaf3 chromo domain contains a unique 38-residue insertion that folds into two short beta-strands and a long flexible loop to flank the beta-barrel core. Both isothermal titration calorimetry and surface plasmon resonance studies indicate that the interaction between the Eaf3 chromo domain and the trimethylated H3K36 peptide is relatively weak, with a K(D) of approximately 10(-4) m. NMR titration studies demonstrate that the methylated H3K36 peptide is bound to the cleft formed by the C-terminal alpha-helix and the beta-barrel core. Site-directed mutagenesis study and in vitro binding assay results show that the conserved aromatic residues Tyr-23, Tyr-81, Trp-84, and Trp-88, which form a hydrophobic pocket at one end of the beta-barrel, are essential for the binding of the methylated H3K36. These results reveal the molecular mechanism of the recognition and binding of the methylated H3K36 by Eaf3 and provide new insights into the functional roles of the Eaf3 chromo domain.     

Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities

The yeast Fus1p SH3 domain binds to peptides containing the consensus motif, R(S/T)(S/T)SL, which is a sharp contrast to most SH3 domains, which bind to PXXP-containing peptides. Here, we have demonstrated that this domain binds to R(S/T)(S/T)SL-containing peptides derived from two putative in vivo binding partners from yeast proteins, Bnr1p and Ste5p, with K_d values in the low micromolar range. The R(S/T)(S/T)SL consensus motif is necessary, but not sufficient for binding to the Fus1p SH3 domain, as residues lying N-terminal to the consensus motif also play a critical role in the binding reaction. Through mutagenesis studies and comparisons to other SH3 domains, we have discovered that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface, which plays the predominant role in binding for most SH3 domains, is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, we created a version of the Fus1p SH3 domain that binds to a PXXP-containing peptide with extremely high affinity (K_d =  40 nM). Based on our data and analysis, we have clearly delineated two distinct surfaces comprising the typical SH3-domain-binding interface and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3-domain-mediated interaction described in the literature. Within this framework, dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of these two surfaces.     

Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3

Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3. Using in situ immunofluorescence, we demonstrate that methyl-K9 H3 and HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl-K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a K(D) of approximately 100 microM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding. Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail.     

Solution structure of the orphan PABC domain from Saccharomyces cerevisiae poly(A)-binding protein

We have determined the solution structure of the PABC domain from Saccharomyces cerevisiae Pab1p and mapped its peptide-binding site. PABC domains are peptide binding domains found in poly(A)-binding proteins (PABP) and are a subset of HECT-family E3 ubiquitin ligases (also known as hyperplastic discs proteins (HYDs)). In mammals, the PABC domain of PABP functions to recruit several different translation factors to the mRNA poly(A) tail. PABC domains are highly conserved, with high specificity for peptide sequences of roughly 12 residues with conserved alanine, phenylalanine, and proline residues at positions 7, 10, and 12. Compared with human PABP, the yeast PABC domain is missing the first alpha helix, contains two extra amino acids between helices 2 and 3, and has a strongly bent C-terminal helix. These give rise to unique peptide binding specificity wherein yeast PABC binds peptides from Paip2 and RF3 but not Paip1. Mapping of the peptide-binding site reveals that the bend in the C-terminal helix disrupts binding interactions with the N terminus of peptide ligands and leads to greatly reduced binding affinity for the peptides tested. No high affinity or natural binding partners from S. cerevisiae could be identified by sequence analysis of known PABC ligands. Comparison of the three known PABC structures shows that the features responsible for peptide binding are highly conserved and responsible for the distinct but overlapping binding specificities.     

Kinetics of Src homology 3 domain association with the proline-rich domain of dynamins: specificity, occlusion, and the effects of phosphorylation

Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.     

Crystal structure of the yeast Phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 A crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the beta-sheet and alpha-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.     

Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein.

The B class cell-attached ephrins mediate contact-dependent cell-cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the second the Src homology 2 (SH2) domain (e.g. the Grb4 adaptor protein). The interaction with Grb4 requires phosphorylation of tyrosine residues on the conserved cytoplasmic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation. However, the exact phosphorylation site(s) required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simultaneously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region (residues 301-333) of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candidate sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide (i.e. ephrinB2(301-333)-pY304) with the SH2 domain. (1)H-(15)N NMR HSQC experiments show that only the ephrinB2(301-333)-pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time, forming a three-component molecular complex. Taken together, our studies identify a novel SH2 domain binding motif, PHpY304EKV, on the cytoplasmic domains of B ephrins that may be essential for reverse signaling via the Grb4 adaptor protein alone or in concert with proteins containing PDZ domains.     

A metadynamic approach to understand the recognition mechanism of the histone H3 tail with the ATRXADD domain

The binding affinity between the histone 3 (H3) tail and the ADD domain of ATRX (ATRX_ADD) increases with the subsequent addition of methyl groups on lysine 9 on H3. To improve our understanding of how the difference in methylation state affects binding between H3 and the ATRX_ADD, we adopted a metadynamic approach to explore the recognition mechanism between the two proteins and identify the key intermolecular interactions that mediate this protein-peptide interaction (PPI). The non-methylated H3 peptide is recognized only by the PHD finger of ATRX_ADD while mono-, di-, and trimethylated H3 is recognized by both the PHD and GATA-like zinc finger of the domain. Furthermore, water molecules play an important role in orienting the lysine 9 anchor towards the GATA-like zinc finger, which results in stabilizing the lysine 9 binding pocket on ATRX_ADD. We compared our computational results against experimentally determined NMR and X-ray structures by demonstrating the RMSD, order parameter S² and hydration number of the complex. The metadynamics data provide new insight into roles of water-bridges and the mechanisms through which K9 hydration stabilizes the H3K9me3:ATRX_ADD PPI, providing context for the high affinity demonstrated between this protein and peptide.     

Mapping the binding site of full length HIV-1 Nef on human Lck SH3 by NMR spectroscopy

Summary The Nef protein of human immunodeficiency virus type 1 (HIV-1) is known to directly bind to the SH3 domain of human lymphocyte specific kinase (Lck) via a proline-rich region located in the amino terminal part of Nef. To address the question whether Nef binding to Lck SH3 involves residues outside the typical poly-proline peptide binding site and whether the Lck unique domain is involved in Nef–Lck interaction, we studied the direct interaction between both molecules using recombinant full-length HIV-1 Nef protein on one side and recombinantly expressed and uniformly ¹⁵N-isotope labeled Lck protein comprising unique and SH3 domains on the other side. Applying nuclear magnetic resonance spectroscopy we could show that only residues of Lck SH3, that are typically involved in binding poly-proline peptides, are affected by Nef binding. Further, for the first time we could rule out that residues of Lck unique domain are involved in binding to full length Nef protein. Thus, interactions of Lck unique domain to cellular partners e.g. CD4 or CD8, are not necessarily competitive with Lck binding to HIV-1 Nef.     

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.     

Domain movement within a gene: a novel evolutionary mechanism for protein diversification

A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.     

Acan125 binding to the SH3 domain of acanthamoeba myosin-IC

The domain organization of Acanthamoeba myosin-I, an oligomodular motor protein, includes a potentially important protein interaction module that is mostly uncharacterized. The Src homology 3, SH3, domain of myosin-I binds Acan125, a protein containing at least two consensus ligand binding domains: C-terminal SH3 binding motifs (PXXP) and N-terminal leucine-rich repeats. We report the first affinities determined for an SH3 domain of any myosin, namely, K(d) = 7 microM for a 21-residue synthetic peptide based on the PXXP domain sequence and K(d) = 0.15 microM for the PXXP domain included in the C-terminus of Acan125. These values are consistent with affinities reported for peptides and proteins that associate with SH3. By deletional analysis we show that only the PXXP domain is required for Acan125 to interact with the SH3 domain of Acanthamoeba myosin-IC (AmyoC(SH3)). The synthetic peptide described above at a concentration near the K(d) for SH3 binding blocked the interaction between native AmyoC and Acan125, mapping the interaction to the PXXP domain of Acan125 and the SH3 domain of myosin-I. These results are consistent with prototypical SH3 binding and suggest that a PXXP module is both necessary and sufficient to interact with an SH3 module of myosin-I.