High resolution structure of the HDGF PWWP domain: a potential DNA binding domain

Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear-targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70-amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in chromatin remodeling. We have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J-couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C-terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a nonspecific DNA-binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA.     

Structural variation in PWWP domains

The PWWP domain is a ubiquitous eukaryotic protein module characterised by a region of sequence similarity of approximately 80 amino acids containing a highly conserved PWWP motif. It is frequently found in proteins associated with chromatin. We have determined the structure of a PWWP domain from the S. pombe protein SPBC215.07c using NMR spectroscopy. The structure is composed of a five stranded beta barrel followed by two alpha helices. Comparison to the recently reported structure of a homologous domain from the mammalian DNA methyltransferase Dnmt3b reveals substantial differences both in the C-terminal helical region and in the PWWP motif.     

The PWWP domain of Dnmt3a and Dnmt3b is required for directing DNA methylation to the major satellite repeats at pericentric heterochromatin

Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.     

Structural and histone binding ability characterizations of human PWWP domains

Background

The PWWP domain was first identified as a structural motif of 100–130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain ‘Royal Family’, which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently.

Methodology/Principal Findings

The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other ‘Royal Family’ members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3.

Conclusions

PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

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Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

The PWWP domain is a weakly conserved sequence motif found in > 60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223--357) has been structurally characterized at 1.8 A resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Delta218 and Delta369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Delta218 protein, which includes the PWWP domain, binds DNA more strongly than Delta369, which lacks the PWWP domain.     

A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus

Hepatoma-derived growth factor (HDGF) and HDGF-related proteins (HRP) belong to a gene family with a well-conserved amino acid sequence at the N-terminus (the hath region). A new member of the HDGF family in humans and mice was identified and cloned; we call it HRP-3. The deduced amino acid sequence from HRP-3 cDNA contained 203 amino acids without a signal peptide for secretion. HRP-3 has its 97-amino-acid sequence at the N-terminus, which is highly conserved with the hath region of the HDGF family proteins. It also has a putative bipartite nuclear localizing signal (NLS) sequence in a similar location in its self-specific region of HDGF and HRP-1. Northern blot analysis shows that HRP-3 is expressed predominantly in the testis and brain, to an intermediate extent in the heart, and to a slight extent in the ovaries, kidneys, spleen, and liver in humans. Transfection of green fluorescent protein (GFP)-tagged HRP-3 cDNA showed that HRP-3 translocated to the nucleus of 293 cells. GFP-HRP-3 transfectants significantly increased their DNA synthesis more than cells transfected with vector only. The HRP-3 gene was mapped to chromosome 15, region q25 by FISH analysis. These findings suggest that a new member of the HDGF gene family, HRP-3, may function mainly in the nucleus of the brain, testis, and heart, probably for cell proliferation.     

Structure of the archaeal translation initiation factor aIF2 beta from Methanobacterium thermoautotrophicum: implications for translation initiation

aIF2 beta is the archaeal homolog of eIF2 beta, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNA(i)(Met) to the 40S ribosomal subunit. We have determined the solution structure of the intact beta-subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 beta is composed of an unfolded N terminus, a mixed alpha/beta core domain and a C-terminal zinc finger. NMR data shows the two folded domains display restricted mobility with respect to each other. Analysis of the aIF2 gamma structure docked to tRNA allowed the identification of a putative binding site for the beta-subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.     

Significance of heparin binding to basic residues in homologous to the amino terminus of hepatoma-derived growth factor and related proteins

Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (K(D)) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.     

The interaction of beta(2)-glycoprotein I domain V with chaperonin GroEL: the similarity with the domain V and membrane interaction

To clarify the mechanism of interaction between chaperonin GroEL and substrate proteins, we studied the conformational changes; of the fifth domain of human beta(2)-glycoprotein I upon binding to GroEL. The fifth domain has a large flexible loop, containing several hydrophobic residues surrounded by positively charged residues, which has been proposed to be responsible for the binding of beta(2)-glycoprotein I to negatively charged phospholipid membranes. The reduction by dithiothreitol of the three intramolecular disulfide bonds of the fifth domain was accelerated in the presence of stoichiometric amounts of GroEL, indicating that the fifth domain was destabilized upon interaction with GroEL. To clarify the GroEL-induced destabilization at the atomic level, we performed H/(2)H exchange of amide protons using heteronuclear NMR spectroscopy. The presence of GroEL promoted the H/(2)H exchange of most of the protected amide protons, suggesting that, although the flexible loop of the fifth domain is likely to be responsible for the initiation of binding to GroEL, the interaction with GroEL destabilizes the overall conformation of the fifth domain.     

Species replacement along a linear coastal habitat: phylogeography and speciation in the red alga Mazzaella laminarioides along the south east pacific

Background

Conserved domains are recognized as the building blocks of eukaryotic proteins. Domains showing a tendency to occur in diverse combinations (??promiscuous?? domains) are involved in versatile architectures in proteins with different functions. Current models, based on global-level analyses of domain combinations in multiple genomes, have suggested that the propensity of some domains to associate with other domains in high-level architectures increases with organismal complexity. Alternative models using domain-based phylogenetic trees propose that domains have become promiscuous independently in different lineages through convergent evolution and are, thus, random with no functional or structural preferences. Here we test whether complex protein architectures have occurred by accretion from simpler systems and whether the appearance of multidomain combinations parallels organismal complexity. As a model, we analyze the modular evolution of the PWWP domain and ask whether its appearance in combinations with other domains into multidomain architectures is linked with the occurrence of more complex life-forms. Whether high-level combinations of domains are conserved and transmitted as stable units (cassettes) through evolution is examined in the genomes of plant or metazoan species selected for their established position in the evolution of the respective lineages.

Results

Using the domain-tree approach, we analyze the evolutionary origins and distribution patterns of the promiscuous PWWP domain to understand the principles of its modular evolution and its existence in combination with other domains in higher-level protein architectures. We found that as a single module the PWWP domain occurs only in proteins with a limited, mainly, species-specific distribution. Earlier, it was suggested that domain promiscuity is a fast-changing (volatile) feature shaped by natural selection and that only a few domains retain their promiscuity status throughout evolution. In contrast, our data show that most of the multidomain PWWP combinations in extant multicellular organisms (humans or land plants) are present in their unicellular ancestral relatives suggesting they have been transmitted through evolution as conserved linear arrangements (??cassettes??). Among the most interesting biologically relevant results is the finding that the genes of the two plant Trithorax family subgroups (ATX1/2 and ATX3/4/5) have different phylogenetic origins. The two subgroups occur together in the earliest land plants Physcomitrella patens and Selaginella moellendorffii.

Conclusion

Gain/loss of a single PWWP domain is observed throughout evolution reflecting dynamic lineage- or species-specific events. In contrast, higher-level protein architectures involving the PWWP domain have survived as stable arrangements driven by evolutionary descent. The association of PWWP domains with the DNA methyltransferases in O. tauri and in the metazoan lineage seems to have occurred independently consistent with convergent evolution. Our results do not support models wherein more complex protein architectures involving the PWWP domain occur with the appearance of more evolutionarily advanced life forms.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor

Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.     

The C terminus of the nuclear protein NuMA: phylogenetic distribution and structure

The C terminus of the nuclear protein NuMA, NuMA-CT, has a well-known function in mitosis via its proximal segment, but it seems also involved in the control of differentiation. To further investigate the structure and function of NuMA, we exploited established computational techniques and tools to collate and characterize proteins with regions similar to the distal portion of NuMA-CT (NuMA-CTDP). The phylogenetic distribution of NuMA-CTDP was examined by PSI-BLAST- and TBLASTN-based analysis of genome and protein sequence databases. Proteins and open reading frames with a NuMA-CTDP-like region were found in a diverse set of vertebrate species including mammals, birds, amphibia, and early teleost fish. The potential structure of NuMA-CTDP was investigated by searching a database of protein sequences of known three-dimensional structure with a hidden Markov model (HMM) estimated using representative (human, frog, chicken, and pufferfish) sequences. The two highest scoring sequences that aligned to the HMM were the extracellular domains of beta3-integrin and Her2, suggesting that NuMA-CTDP may have a primarily beta fold structure. These data indicate that NuMA-CTDP may represent an important functional sequence conserved in vertebrates, where it may act as a receptor to coordinate cellular events.     

Structure-based mechanism of ligand binding for periplasmic solute-binding protein of the Bug family

Bug proteins form a large family of periplasmic solute-binding proteins well represented in beta-proteobacteria. They adopt a characteristic Venus flytrap fold with two globular domains bisected by a ligand-binding cleft. The structures of two liganded Bug proteins have revealed that the family is specific for carboxylated solutes, with a characteristic mode of binding involving two highly conserved beta strand-beta turn-alpha helix motifs originating from each domain. These two motifs form hydrogen bonds with a carboxylate group of the ligand, both directly and via conserved water molecules, and have thus been termed the carboxylate pincers. In both crystallized Bug proteins, the ligands were found enclosed between the two domains and inaccessible to solvent, suggesting an inter-domain hinge-bending motion upon ligand binding. We report here the first structures of an open, unliganded Bug protein and of the same protein with a citrate ion bound in the open cavity. One of the ligand carboxylate groups is bound to one half of the carboxylate pincers by the beta strand-beta turn-alpha helix motif from domain 1, and the citrate ion forms several additional interactions with domain 1. The ligand is accessible to solvent and has very few contacts with domain 2. In this open, liganded structure, the second part of the carboxylate pincers originating from domain 2 is not stabilized by ligand binding, and a loop replaces the beta turn. In the unliganded structure, both motifs of the carboxylate pincers are highly mobile, and neither of the two beta turns is formed. Thus, ligand recognition is performed by domain 1, with the carboxylate group serving as an initial anchoring point. Stabilization of the closed conformation requires proper interactions to be established with domain 2, and thus domain 2 discriminates between productively and non-productively bound ligands.