Tarun Gheyi

Crystal structure of the human PRMT5:MEP50 complex

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 β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.

Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.

The 2.5 Å crystal structure of the SIRT1 catalytic domain bound to nicotinamide adenine dinucleotide (NAD+) and an indole (EX527 analogue) reveals a novel mechanism of histone deacetylase inhibition.

The sirtuin SIRT1 is a NAD(+)-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD(+) and the 27 analogue compound 35. 35 binds deep in the catalytic cleft, displacing the NAD(+) nicotinamide and forcing the cofactor into an extended conformation. The extended NAD(+) conformation sterically prevents substrate binding. The SIRT1/NAD(+)/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.

Structures of a minimal human CFTR first nucleotide- binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

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.

Structural Context of Disease-Associated Mutations and Putative Mechanism of Autoinhibition Revealed by X-Ray Crystallographic Analysis of the EZH2-SET Domain.

The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.

LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2

Background: SMYD2 is a methyltransferase whose role in cancer is poorly understood and is lacking cell-active chemical tools. Results: We describe LLY-507, a small molecule inhibitor of SMYD2. Conclusion: LLY-507 is potent, selective, cell-active, and binds SMYD2 in a high resolution co-crystal. Significance: LLY-507 is a first-in-class cell-potent chemical probe that will be valuable in dissecting SMYD2 biology. 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 Lys370 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.

High Throughput Protein Production and Crystallization at NYSGXRC

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.

Structural Bases of PAS Domain-regulated Kinase (PASK) Activation in the Absence of Activation Loop Phosphorylation

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.

Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex

Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.

Mass spectrometry guided in situ proteolysis to obtain crystals for X-ray structure determination.

A strategy for increasing the efficiency of protein crystallization/structure determination with mass spectrometry has been developed. This approach combines insights from limited proteolysis/mass spectrometry and crystallization via in situ proteolysis. The procedure seeks to identify protease-resistant polypeptide chain segments from purified proteins on the time-scale of crystal formation, and subsequently crystallizing the target protein in the presence of the optimal protease at the right relative concentration. We report our experience with 10 proteins of unknown structure, two of which yielded high-resolution X-ray structures. The advantage of this approach comes from its ability to select only those structure determination candidates that are likely to benefit from application of in situ proteolysis, using conditions most likely to result in formation of a stable proteolytic digestion product suitable for crystallization.