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1.
J Biol Chem ; 290(22): 13641-53, 2015 May 29.
Article in English | MEDLINE | ID: mdl-25825497

ABSTRACT

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.


Subject(s)
Antineoplastic Agents/chemistry , Benzamides/chemistry , Enzyme Inhibitors/chemistry , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Neoplasms/enzymology , Pyrrolidines/chemistry , Cell Line, Tumor , Cell Proliferation , Chromatin/chemistry , Computational Biology , Crystallization , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Epigenesis, Genetic , Histones/chemistry , Humans , Mass Spectrometry , Neoplasms/drug therapy , Peptides/chemistry , Protein Denaturation , Proteomics , Tumor Suppressor Protein p53/metabolism
2.
Proc Natl Acad Sci U S A ; 109(44): 17960-5, 2012 Oct 30.
Article in English | MEDLINE | ID: mdl-23071334

ABSTRACT

Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Protein-Arginine N-Methyltransferases/chemistry , Catalytic Domain , Crystallography, X-Ray , Dimerization , Humans , Models, Molecular , Protein Conformation
3.
Protein Sci ; 20(12): 2080-94, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21998098

ABSTRACT

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.


Subject(s)
Aminohydrolases/metabolism , Catalase/metabolism , Escherichia coli/enzymology , Iron/metabolism , Aminohydrolases/chemistry , Aminohydrolases/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Hydrogen Peroxide/metabolism , Hydroxyl Radical/metabolism , Iron/chemistry , Models, Molecular , Mutagenesis , Oxidation-Reduction , Superoxides/metabolism
4.
PLoS One ; 6(7): e22290, 2011.
Article in English | MEDLINE | ID: mdl-21779408

ABSTRACT

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.


Subject(s)
Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/metabolism , Adenosine/analogs & derivatives , Adenosine/metabolism , Binding Sites , Histones/metabolism , Humans , Male , Protein Binding , Protein Structure, Secondary , Structure-Activity Relationship
5.
J Biol Chem ; 285(52): 41034-43, 2010 Dec 24.
Article in English | MEDLINE | ID: mdl-20943661

ABSTRACT

Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.


Subject(s)
Protein Serine-Threonine Kinases/chemistry , Enzyme Activation/physiology , Humans , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship
6.
Protein Sci ; 19(10): 1917-31, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20687133

ABSTRACT

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T(m)) by 6-7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) <==> I(T)(±MgATP) → A(T) → (A(T))(n). The equilibrium unfolding to intermediate, I(T), is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A(T), for which aggregation to (A(T))(n) and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A(T).


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Mutation , Nucleotides/chemistry , Protein Folding , Algorithms , Binding Sites/genetics , Calorimetry, Differential Scanning , Circular Dichroism , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Kinetics , Nucleotides/metabolism , Phenylalanine/genetics , Protein Binding , Protein Denaturation , Protein Stability , Protein Structure, Tertiary , Sequence Deletion , Thermodynamics , Transition Temperature
7.
Protein Eng Des Sel ; 23(5): 375-84, 2010 May.
Article in English | MEDLINE | ID: mdl-20150177

ABSTRACT

Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Models, Molecular , Protein Conformation , Protein Structure, Tertiary/genetics , Binding Sites/genetics , Cloning, Molecular , Crystallization , Cystic Fibrosis Transmembrane Conductance Regulator/isolation & purification , DNA Primers/genetics , Dimerization , Humans , Mutation/genetics
8.
Mol Cancer Ther ; 8(12): 3181-90, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19934279

ABSTRACT

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.


Subject(s)
Adenosine Triphosphate/pharmacology , Neoplasms/prevention & control , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridazines/pharmacology , Triazoles/pharmacology , Xenograft Model Antitumor Assays , Animals , Catalysis/drug effects , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Dose-Response Relationship, Drug , Female , Humans , Kinetics , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation/drug effects , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-met/chemistry , Proto-Oncogene Proteins c-met/metabolism , Pyridazines/chemistry , Triazoles/chemistry , Tumor Burden/drug effects
9.
Methods Mol Biol ; 426: 561-75, 2008.
Article in English | MEDLINE | ID: mdl-18542890

ABSTRACT

Phase II of the Protein Structure Initiative, funded by the NIH NIGMS (National Institute of General Medical Sciences), is a 5-year effort to determine thousands of protein structures. The New York SGX Research Center for Structural Genomics (NYSGXRC) is one of the four large-scale production centers tasked with determining 100-200 structures annually. Almost all protein production is carried out using the high throughput structural biology platform at SGX Pharmaceuticals (SGX), which supplies 120 or more ultrapure proteins per month for NYSGXRC crystallization and structure determination activities. Protocols for PCR, cloning, expression/solubility testing, fermentation, purification, and crystallization are described. General protocols and detailed experimental results for each target are updated weekly at the public PepcDB website (pepcdb.pdb.org/), and all NYSGXRC clones should be available in 2008 through the PlasmID resource operated by the Harvard Institute of Proteomics.


Subject(s)
Proteins/chemistry , Proteins/isolation & purification , Proteomics/methods , Proteomics/organization & administration , Cloning, Molecular/methods , Crystallography, X-Ray/methods , New York City , Polymerase Chain Reaction/methods , Proteins/genetics
10.
J Struct Funct Genomics ; 8(2-3): 121-40, 2007 Sep.
Article in English | MEDLINE | ID: mdl-18058037

ABSTRACT

The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.


Subject(s)
Genomics , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/genetics , Animals , Crystallography, X-Ray , Humans , Multigene Family , Phosphoprotein Phosphatases/classification , Phosphoprotein Phosphatases/physiology , Sequence Analysis, DNA
11.
J Struct Funct Genomics ; 6(2-3): 225-32, 2005.
Article in English | MEDLINE | ID: mdl-16211523

ABSTRACT

Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.


Subject(s)
Multi-Institutional Systems/organization & administration , Proteins/chemistry , Proteins/metabolism , Proteomics/methods , Proteomics/organization & administration , Cloning, Molecular/methods , Crystallography, X-Ray/methods , New York City , Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/isolation & purification
12.
J Biol Chem ; 280(2): 1346-53, 2005 Jan 14.
Article in English | MEDLINE | ID: mdl-15528182

ABSTRACT

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Nucleotides/metabolism , Protein Folding , Sequence Deletion/genetics , Amino Acid Sequence , Binding Sites , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Denaturation , Protein Renaturation , Protein Structure, Tertiary , Solubility
13.
EMBO J ; 23(2): 282-93, 2004 Jan 28.
Article in English | MEDLINE | ID: mdl-14685259

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.


Subject(s)
Adenosine Triphosphate/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Models, Molecular , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Mice , Molecular Sequence Data , Mutation , Phosphorylation , Protein Structure, Tertiary , Sequence Alignment
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