Molecular Basis For Specific Recognition Research Articles

Uncovering the mechanistic basis for specific recognition of monomethylated H3K4 by the CW domain of Arabidopsis histone methyltransferase SDG8

Chromatin consists of DNA and histones, and specific histone modifications that determine chromatin structure and activity are regulated by three types of proteins, called writer, reader, and eraser. Histone reader proteins from vertebrates, vertebrate-infecting parasites, and higher plants possess a CW domain, which has been reported to read histone H3 lysine 4 (H3K4). The CW domain of Arabidopsis SDG8 (also called ASHH2), a histone H3 lysine 36 methyltransferase, preferentially binds monomethylated H3K4 (H3K4me1), unlike the mammalian CW domain protein, which binds trimethylated H3K4 (H3K4me3). However, the molecular basis of the selective binding by the CW domain of SDG8 (SDG8-CW) remains unclear. Here, we solved the 1.6-Å-resolution structure of SDG8-CW in complex with H3K4me1, which revealed that residues in the C-terminal α-helix of SDG8-CW determine binding specificity for low methylation levels at H3K4. Moreover, substitutions of key residues, specifically Ile-915 and Asn-916, converted SDG8-CW binding preference from H3K4me1 to H3K4me3. Sequence alignment and mutagenesis studies revealed that the CW domain of SDG725, the homolog of SDG8 in rice, shares the same binding preference with SDG8-CW, indicating that preference for low methylated H3K4 by the CW domain of ASHH2 homologs is conserved among higher-order plants. Our findings provide first structural insights into the molecular basis for specific recognition of monomethylated H3K4 by the H3K4me1 reader protein SDG8 from Arabidopsis.

Abstract 5232: The molecular basis for specific recognition of the biologically relevant hybrid-2 type human telomeric G-quadruplex by epiberberine

Abstract G-quadruplex structures have been shown to form in human telomeres, and the formation of G-quadruplexes can inhibit telomerase, which plays a key role in cancers by stabilizing telomere length and integrity thereby granting limitless replicative potential. The human telomeric G-quadruplex is thus considered to be a potential target for cancer therapeutics. In physiologically relevant potassium solution, human telomeric DNA sequences form two equilibrating hybrid-type G-quadruplex structures, with the hybrid-2 structure being the predominant form in extended sequences. We discovered that epiberberine (EPI), a naturally occurring protoberberine alkaloid, can specifically bind the hybrid-2 human telomeric G-quadruplex and can induce the conversion to the hybrid-2 structure. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of the 1:1 complex of EPI and hybrid-2 human telomeric G-quadruplex. Our NMR structure shows EPI bound at the 5’ end of the hybrid-2 telomeric quadruplex with an unexpectedly large drug-induced conformational change in the flanking and loop regions, creating a very well-defined drug binding pocket with extensive capping structures. The EPI molecule and 5’ flanking adenine form an induced quasi-triad plane which is intercalated between the external 5’ tetrad and capping structures. Two layers of new capping structures are formed with the 5’ flanking segment and the second TTA lateral loop to cover the “intercalated quasi-triad”. Notably, the well-defined EPI-induced multi-layer binding-site arrangement is only possible in the hybrid-2 folding topology of telomeric DNA. Our results provide important insights into specific targeting of the physiologically relevant hybrid-2 human telomeric G-quadruplex by a small molecule compound; EPI’s asymmetric crescent-shape and the positioning of its dioxolane moiety, as well as the hybrid-2 folding and the loop and flanking bases in the human telomeric DNA sequence, all contribute toward the specific recognition of EPI. In addition, we have conducted polymerase stop assays, which confirmed that EPI can stabilize the human telomeric G-quadruplex to inhibit polymerase activity. Citation Format: Clement Lin, Guanhui Wu, Yong Shao, Danzhou Yang. The molecular basis for specific recognition of the biologically relevant hybrid-2 type human telomeric G-quadruplex by epiberberine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2017-5232

Structural Insights into Substrate Recognition by Clostridium difficile Sortase.

Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26–PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB.

Molecular Basis for Specific Recognition of Bacterial Ligands by NAIP/NLRC4 Inflammasomes

NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NLR apoptosis inhibitory protein (NAIP) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLR family, CARD-containing 4 (NLRC4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes.

Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF

Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.

The Poxvirus p28 Virulence Factor Is an E3 Ubiquitin Ligase

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.

Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding.

The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (SXL), which has two RRMs, can directly compete with the essential splicing factor U2AF(65), which has three RRMs, for binding to specific Py tracts. We have combined site-specific photocross-linking and chemical cleavage of the proteins to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF(65) shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF(65) and SXL can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. We present a model to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity.

Conformation and dynamics of normal and damaged DNA.

The genetic information that determines the structure and function of living organisms is encoded in the nucleotide sequence of double-stranded DNA molecules. Despite an apparent structural homogeneity displayed by DNA, subtle local variations in structure and dynamics are functionally significant. Short sequences exhibit specificity for regulatory and catalytic proteins, which mediate fundamental processes necessary to the survival of the cell. However, the molecular basis for specific recognition is still incompletely understood. The "indirect readout" mechanism suggests that the relative propensity of DNA to undergo structural deformations induced by the protein contributes to specific protein-DNA recognition. Although the hypothesis was originally formulated to explain recognition of specific nucleic acid sequences by DNA-binding proteins, it may have particular application to the recognition of DNA damage, because damaged sites in DNA have different equilibrium structure and dynamics from undamaged DNA. In this work, we review the approaches that we took to investigate the questions of sequence- and damage-dependent structure and dynamics of DNA. We describe a statistical thermodynamic model that relates DNA configurational flexibility to sequence-specific protein-DNA binding. The model provides a theoretical basis for interpreting experimental measurements of DNA dynamics. We describe results from MCSCF calculations of the excited states of 2-aminopurine (2AP), which provide the theoretical basis for the intramolecular mechanism of quenching as well as the effect of environment on this process. We then describe our investigations of the effect of stacking, base pairing, and base dynamics on the fluorescence of 2-AP in model systems, which allow us to develop the relationships between steady-state and time-resolved fluorescence parameters on the one hand and local structural and dynamic properties of DNA on the other hand. Finally, we describe the application of the experimental approach to study the conformational heterogeneity of DNA abasic sites, a commonly occurring type of DNA damage. We demonstrate the power of the experimental algorithm to characterize the physical differences between undamaged and damaged DNA, as well as the effects of nucleic acid sequence in both of these contexts. Thus, the work described herein comprises a combination of theoretical and experimental approaches to the problem of sequence- and damage-dependent DNA deformation.

Dynamic Features of the Subunit Interface of Cu,Zn Superoxide Dismutase as Probed by Tryptophan Phosphorescence

As part of the more general inquiry on the molecular basis of specific recognition between macromolecules, the subunit–subunit interface structure of dimeric superoxide dismutase from Photobacterium leiognathi has been probed selectively by the phosphorescence emission of Trp-73, located at the subunit contact region. Copper at the catalytic site was found to quench completely the delayed emission and therefore all studies were conducted with the copper-free or Cd2+-substituted protein. The spectrum at 140 K is diagnostic for an indole ring located in a hydrophobic environment whereas a degree of spectral broadening indicates that the local structure is not unique. Environmental heterogeneity is confirmed by the nonuniform phosphorescence decay in buffer, at 274 K, with lifetime components of 44 and 20 ms of practically equal amplitude. Information on the flexibility of the interface region was gathered from both the intrinsic lifetime and the accessibility of acrylamide to the site of the chromophore. The magnitude of the intrinsic lifetime, its temperature dependence, and the accessibility to solutes like acrylamide describe a tight dimeric structure in which hydrophobic interactions seem to play an important role. In particular the acrylamide bimolecular rate constant is 1.4 × 104 M−1 s−1 and indicates highly hindered diffusion of the solute through the interface region. Cd2+ complexation to the apoprotein caused no detectable changes in protein conformation although the metal was able to influence the flexibility of the Trp-73 environment, indicating the occurrence of a long-range communication between the intersubunit surface and the active site, which is more than 16 Å away.

The effects of N7-methylguanine on duplex DNA structure

Background: Non-enzymatic methylation of DNA by endogenous and exogenous agents produces a variety of adducts, of which the predominant one is N7-methyl-2′-deoxyguanosine (m 7dG).Although it is known that living organisms counter the deleterious effects of m 7dG by producing adduct-specific DNA repair proteins, the molecular basis for specific recognition and catalysis by these proteins is poorly understood. In addition to its role as an endogenous DNA adduct, m 7dG is also widely used as an in vitro probe of protein-DNA interactions. We set out to examine whether incorporation of m 7dG into DNA affects duplex DNA structure. Results: We carried out a large-scale synthesis of a dodecamer containing the m 7dG adduct at a single, defined position. Because the instability of m 7dG precludes its incorporation into oligonucleotides by standard solid-phase methods, a novel strategy employing chemical and enzymatic synthesis was used. Characterization of the m 7G-containing dodecamer by NMR reveals no structural distortion; indeed, m 7dG appears to encourage a modest shift toward a more characteristically B-form duplex. Conclusions: These results argue strongly against induced DNA distortion as a mechanism for specific recognition of m 77dG by adduct-specific repair proteins. The broad substrate specificity of these repair proteins disfavors a model involving direct recognition of aberrantly placed methyl groups; hence, it may be that m 7dG is recognized indirectly, perhaps by its effects on the dynamics of DNA. On the other hand, the evidence presented here suggests that m 7dG interferes directly with sequence-specific recognition by DNA-binding proteins by steric blockage or by masking of required contact functionalities. The synthetic methodology used here should be generally applicable to high-resolution structural studies of oligonucleotides bearing adducts that are unstable to the conditions of solid-phase DNA synthesis.

Molecular Basis for Specific Recognition of Both RNA and DNA by a Zinc Finger Protein

Transcription factor IIIA (TFIIIA) from Xenopus oocytes binds both the internal control region of the 5S ribosomal RNA genes and the 5S RNA transcript itself. The nucleic acid binding domain of TFIIIA contains nine tandemly repeated zinc finger motifs. A series of precisely truncated forms of this protein have been constructed and assayed for 5S RNA and DNA binding. Different sets of zinc fingers were found to be responsible for high affinity interactions with RNA and with DNA. These results explain how a single protein can exhibit equal affinities for these two very different nucleic acids.