I-TASSER server: new development for protein structure and function predictions

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumorsuppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.Cancer is increasingly characterized by alterations in chromatin-modifying enzymes (1). SETD2, a non-redundant histone H3 lysine 36 (H3K36) 4 methyltransferase (2), has been found to be mutated in a growing list of tumor types, most notably in clear cell renal cell carcinoma (ccRCC) (1, 3, 4), but also in high grade gliomas (5), breast cancer (6), bladder cancer (7), and acute lymphoblastic leukemia (8 -10). Recent studies exploring intratumor heterogeneity in ccRCC identified distinct mutations in SETD2 from spatially distinct subsections of an individual tumor, suggesting that mutation of SETD2 is a critical and selected event in ccRCC cancer progression (11). Mutations in SETD2 are predominantly inactivating, such as early nonsense or frameshift mutations, which lead to nonfunctional protein and global loss of H3K36 trimethylation (H3K36me3) (4,11,12). Missense mutations tend to cluster in two domains (1,4,12,13): the SET domain, which catalyzes H3K36me3 (14), and the Set2 Rpb1 interaction (SRI) domain, which mediates the interaction between SETD2 and the hyperphosphorylated form of RNA polymerase II (RNAPII) (13).SETD2 and its yeast counterpart, Set2, both associate with RNAPII in a co-transcriptional manner (13,15,16). In yeast, Set2 mediates all H3K36 methylation states (H3K36me1/me2/ me3) (17) and regulates the recruitment of chromatin-remodeling enzymes (Isw1b) and a histone deacetylase (Rpd3) (18) that functions to keep gene bodies deacetylated, thereby maintaining a more compact chromatin structure (19,20) that is more resistant to inappropr...

Section: Setd2 and Set2 Share A High Degree Of Structural And Sequencmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

Hacker

Fahey

Shinsky

et al. 2016

“…We used the x-ray crystal structure of the Yfh1 Y73A trimer with the entire N-terminal region (residues 52-172) resolved (30) (PDB code 3OEQ). Essentially identical homology models of the Isu1 monomer were generated by the I-TASSER web resource (42) and by Phyre2 using the NMR structure of H. influenzae IscU, and the I-TASSER model was then used for docking.…”

Section: Docking Of Yfh1mentioning

confidence: 99%

“…Nfs1 Modeling-The homology model for yeast Nfs1 was created using I-TASSER (42), and the model with the highest confidence score (Ϫ1.14) was chosen. Pyridoxal 5Ј-phosphate (PLP) and [2Fe-2S] cluster were modeled based on the A. fulgi-…”

Section: Docking Of Yfh1mentioning

confidence: 99%

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

Ranatunga

Gakh

Galeano

et al. 2016

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.

“…I-TASSER Structural Prediction and TAF2 Site-directed Mutagenesis-Primary amino acid sequences for S. cerevisiae (yeast) Taf2, Drosophila melanogaster (fly) Taf2, and Homo sapiens (human) Taf2 were submitted to the I-TASSER server for 3D structure prediction (72). Resulting 3D models of yeast, fly, and human were imported in PyMOL, displayed in cartoon format and colored based on secondary structure.…”

Section: Taf2 Molecular Genetic Dissection and Taf14 Interactionmentioning

confidence: 99%

The C Terminus of the RNA Polymerase II Transcription Factor IID (TFIID) Subunit Taf2 Mediates Stable Association of Subunit Taf14 into the Yeast TFIID Complex

Feigerle

Weil

2016

The evolutionarily conserved RNA polymerase II transcription factor D (TFIID) complex is composed of TATA box-binding protein (TBP) and 13 TBP-associated factors (Tafs). The mechanisms by which many Taf subunits contribute to the essential function of TFIID are only poorly understood. To address this gap in knowledge, we present the results of a molecular genetic dissection of the TFIID subunit Taf2. Through systematic site-directed mutagenesis, we have discovered 12 taf2 temperature-sensitive (ts) alleles. Two of these alleles display growth defects that can be strongly suppressed by overexpression of the yeast-specific TFIID subunit TAF14 but not by overexpression of any other TFIID subunit. In Saccharomyces cerevisiae, Taf14 is also a constituent of six other transcription-related complexes, making interpretation of its role in each of these complexes difficult. Although Taf14 is not conserved as a TFIID subunit in metazoans, it is conserved through its chromatinbinding YEATS domain. Based on the Taf2-Taf14 genetic interaction, we demonstrate that Taf2 and Taf14 directly interact and mapped the Taf2-Taf14 interaction domains. We used this information to identify a Taf2 separation-of-function variant (Taf2-⌬C). Although Taf2-⌬C no longer interacts with Taf14 in vivo or in vitro, it stably incorporates into the TFIID complex. In addition, purified Taf2-⌬C mutant TFIID is devoid of Taf14, making this variant a powerful reagent for determining the role of Taf14 in TFIID function. Furthermore, we characterized the mechanism through which Taf14 suppresses taf2 ts alleles, shedding light on how Taf2-Taf14 interaction contributes to TFIID complex organization and identifying a potential role for Taf14 in mediating TFIID-chromatin interactions.RNA polymerase II and the general RNA polymerase II transcription factors (TFIIA,