Chemical Biology Approaches for Characterization of Epigenetic Regulators.

Chemical biology approaches are a powerful means to functionally characterize epigenetic regulators such as histone modifying enzymes. We outline experimental protocols and best practices for the cellular characterization and use of "chemical probes" that selectively inhibit protein methyltransferases, many of which methylate histones to regulate heritable gene expression patterns. We describe biomarker assays to validate the probes in specific cellular systems, and provide guidelines for their use in functional characterization of methyltransferases including detailed protocols, examples, and controls. Together these techniques enable precision manipulation of cellular epigenomes and the exploration of the therapeutic potential of epigenetic targets in human disease.

Optimization of resolution and sensitivity of 4D NOESY using multi-dimensional decomposition.

Highly resolved multi-dimensional NOE data are essential for rapid and accurate determination of spatial protein structures such as in structural genomics projects. Four-dimensional spectra contain almost no spectral overlap inherently present in lower dimensionality spectra and are highly amenable to application of automated routines for spectral resonance location and assignment. However, a high resolution 4D data set using conventional uniform sampling usually requires unacceptably long measurement time. Recently we have reported that the use of non-uniform sampling and multi-dimensional decomposition (MDD) can remedy this problem. Here we validate accuracy and robustness of the method, and demonstrate its usefulness for fully protonated protein samples. The method was applied to 11 kDa protein PA1123 from structural genomics pipeline. A systematic evaluation of spectral reconstructions obtained using 15-100% subsets of the complete reference 4D 1H-13C-13C-1H NOESY spectrum has been performed. With the experimental time saving of up to six times, the resolution and the sensitivity per unit time is shown to be similar to that of the fully recorded spectrum. For the 30% data subset we demonstrate that the intensities in the reconstructed and reference 4D spectra correspond with a correlation coefficient of 0.997 in the full range of spectral amplitudes. Intensities of the strong, middle and weak cross-peaks correlate with coefficients 0.9997, 0.9965, and 0.83. The method does not produce false peaks. 2% of weak peaks lost in the 30% reconstruction is in line with theoretically expected noise increase for the shorter measurement time. Together with good accuracy in the relative line-widths these translate to reliable distance constrains derived from sparsely sampled, high resolution 4D NOESY data.

Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase.

BACKGROUND: The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. RESULTS: The structure of SurE from Thermotoga maritima was determined at 2.0 A. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. CONCLUSIONS: The structure of SurE provided information about the protein's fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

Solution structure and dynamics of yeast elongin C in complex with a von Hippel-Lindau peptide.

Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes. A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein. Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C. Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site. The bound VHL peptide is entirely helical. Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that fits VHL(157-171). Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation. Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition.

Latent and active p53 are identical in conformation.

p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.

SPINE: an integrated tracking database and data mining approach for identifying feasible targets in high-throughput structural proteomics.

High-throughput structural proteomics is expected to generate considerable amounts of data on the progress of structure determination for many proteins. For each protein this includes information about cloning, expression, purification, biophysical characterization and structure determination via NMR spectroscopy or X-ray crystallography. It will be essential to develop specifications and ontologies for standardizing this information to make it amenable to retrospective analysis. To this end we created the SPINE database and analysis system for the Northeast Structural Genomics Consortium. SPINE, which is available at bioinfo.mbb.yale.edu/nesg or nesg.org, is specifically designed to enable distributed scientific collaboration via the Internet. It was designed not just as an information repository but as an active vehicle to standardize proteomics data in a form that would enable systematic data mining. The system features an intuitive user interface for interactive retrieval and modification of expression construct data, query forms designed to track global project progress and external links to many other resources. Currently the database contains experimental data on 985 constructs, of which 740 are drawn from Methanobacterium thermoautotrophicum, 123 from Saccharomyces cerevisiae, 93 from Caenorhabditis elegans and the remainder from other organisms. We developed a comprehensive set of data mining features for each protein, including several related to experimental progress (e.g. expression level, solubility and crystallization) and 42 based on the underlying protein sequence (e.g. amino acid composition, secondary structure and occurrence of low complexity regions). We demonstrate in detail the application of a particular machine learning approach, decision trees, to the tasks of predicting a protein's solubility and propensity to crystallize based on sequence features. We are able to extract a number of key rules from our trees, in particular that soluble proteins tend to have significantly more acidic residues and fewer hydrophobic stretches than insoluble ones. One of the characteristics of proteomics data sets, currently and in the foreseeable future, is their intermediate size ( approximately 500-5000 data points). This creates a number of issues in relation to error estimation. Initially we estimate the overall error in our trees based on standard cross-validation. However, this leaves out a significant fraction of the data in model construction and does not give error estimates on individual rules. Therefore, we present alternative methods to estimate the error in particular rules.

The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold.

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.

Structure and functionality of a designed p53 dimer.

P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.

Protein production: feeding the crystallographers and NMR spectroscopists.

Protein purification efforts for structural genomics will focus on automation for the readily-expressed proteins, and process development for the more difficult ones, such as membrane proteins. Thousands of proteins are expected to be produced in the next few years. The purified proteins will be valuable reagents for the entire research community.

Assignment of 1H(N), 15N, 13C(alpha), 13CO and 13C(beta) resonances in a 67 kDa p53 dimer using 4D-TROSY NMR spectroscopy.

The p53 tumor suppressor is a transcription factor that plays a crucial role in the activation of genes in response to DNA damage. As a first step towards detailed structural studies of the molecule aimed at understanding its regulation, we have used 4D-TROSY triple resonance NMR spectroscopy to obtain nearly complete 1H(N), 15N, 13C(alpha), 13CO and 13C(beta) resonance assignments of a dimeric form of the protein comprising DNA-binding and oligomerization domains (67 kDa). A simple comparison of 4D spectra recorded on p53 molecules consisting of DNA-binding and oligomerization domains with and without the regulatory domain establishes that both constructs have essentially identical chemical shifts. Although the affinity of p53 for target DNA is decreased in constructs containing the regulatory domain, the chemical shift results reported here suggest that this decrease is not due to specific domain interactions involving the regulatory portion of the molecule, or alternatively, that such interactions require the presence of DNA.

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53.

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53 (residues 325-355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the alpha-helices at the dimer-dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum.

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.

DNA binding specificity studies of four ETS proteins support an indirect read-out mechanism of protein-DNA recognition.

Members of the ETS family of transcription factors are involved in several developmental and physiological processes, and, when overexpressed or misexpressed, can contribute to a variety of cancers. Each family member has a conserved DNA-binding domain that recognizes DNA sequences containing a G-G-A trinucleotide. Discrimination between potential ETS-binding sites appears to be governed by both the nucleotides flanking the G-G-A sequence and protein-protein interactions. We have used an adaptation of the "length-encoded multiplex" approach (Desjarlais, J. R., and Berg, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11099-11103) to define DNA binding specificities for four ETS proteins: Fli-1, SAP-1, PU.1, and TEL. Our results support a model in which cooperative effects among neighboring bases flanking the central G-G-A site contribute to the formation of stable ETS/DNA complexes. These results are consistent with a mechanism for specific DNA binding that is partially governed by an indirect read-out of the DNA sequence, in which a sequence-specific DNA conformation is sensed or induced.

Solution structure of the RNA polymerase subunit RPB5 from Methanobacterium thermoautotrophicum.

RPB5 is an essential subunit of eukaryotic and archaeal RNA polymerases. It is a proposed target for transcription activator proteins in eukaryotes, but the mechanism of interaction is not known. We have determined the solution structure of the RPB5 subunit from the thermophilic archeon, Methanobacterium thermoautotrophicum. MtRBP5 contains a four-stranded beta-sheet platform supporting two alpha-helices, one on each side of the beta-sheet, resulting in an overall mushroom shape that does not appear to have any structural homologues in the structural database. The position and conservation of charged surface residues suggests possible modes of interaction with other proteins, as well as a rationale for the thermal stability of this protein.