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The Cbfb gene, the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF), is essential for embryogenesis. Specifically, it is found to be required for CBF(alpha)2 function in vivo. Homozygous mutation of the Cbfb gene is found to result in the same range of abnormalities observed in Cbfa2-deficient mice, including embryonic lethality at midgestation due to hemorrhaging in the central nervous system and inhibition of fetal liver hematopoiesis.

Summary: Full understanding of the mechanism of function of multidomain proteins is dependent on our knowledge of their supramodular architecture in solution. This is a nontrivial task for both X-ray crystallography and NMR, because intrinsic flexibility makes crystallization of these proteins difficult, while their size creates a challenge for NMR. Here, we describe synergistic application of data derived from X-ray crystallography and NMR residual dipolar couplings (RDCs) to address the question of the supramodular structure of a two-domain protein, syntenin. Syntenin is a 32 kDa molecule containing two PDZ domains and is involved in cytoskeleton-membrane organization. We show that the mutual disposition of the PDZ domains clearly differs from that seen in the crystal structure, and we provide evidence that N- and C-terminal fragments of syntenin, hitherto presumed to lack ordered structure, contain folded structural elements in the full-length protein in contact with the PDZ tandem. [Copyright &y& Elsevier]

Summary Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. Nuclear magnetic resonance data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N terminus when bound to Tom1 GAT through high-affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors the release of Tollip from endosomal membranes, allowing Tollip to commit to cargo trafficking. [ABSTRACT FROM AUTHOR]

Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins. [ABSTRACT FROM AUTHOR]

The MLL CXXC domain binds nonmethylated CpG-contain-ing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethyla-tion (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation. [ABSTRACT FROM AUTHOR]

Summary: Mixed lineage leukemia (MLL) fusion proteins cause oncogenic transformation of hematopoietic cells by constitutive recruitment of elongation factors to HOX promoters, resulting in overexpression of target genes. The structural basis of transactivation by MLL fusion partners remains undetermined. We show that the ANC1 homology domain (AHD) of AF9, one of the most common MLL translocation partners, is intrinsically disordered and recruits multiple transcription factors through coupled folding and binding. We determined the structure of the AF9 AHD in complex with the elongation factor AF4 and show that aliphatic residues, which are conserved in each of the AF9 binding partners, form an integral part of the hydrophobic core of the complex. Nuclear magnetic resonance relaxation measurements show that AF9 retains significant dynamic behavior which may facilitate exchange between disordered partners. We propose that AF9 functions as a signaling hub that regulates transcription through dynamic recruitment of cofactors in normal hematopoiesis and in acute leukemia. [Copyright &y& Elsevier]

Summary: Nuclear magnetic resonance paramagnetic relaxation enhancement (PRE) measures long-range distances to isotopically labeled residues, providing useful constraints for protein structure prediction. The method usually requires labor-intensive conjugation of nitroxide labels to multiple locations on the protein, one at a time. Here a computational procedure, based on protein sequence and simple secondary structure models, is presented to facilitate optimal placement of a minimum number of labels needed to determine the correct topology of a helical transmembrane protein. Tests on DsbB (four helices) using just one label lead to correct topology predictions in four of five cases, with the predicted structures <6 Å to the native structure. Benchmark results using simulated PRE data show that we can generally predict the correct topology for five and six to seven helices using two and three labels, respectively, with an average success rate of 76% and structures of similar precision. The results show promise in facilitating experimentally constrained structure prediction of membrane proteins. [ABSTRACT FROM AUTHOR]

Summary: AML1/ETO results from the t(8;21) associated with 12%–15% of acute myeloid leukemia. The AML1/ETO MYND domain mediates interactions with the corepressors SMRT and N-CoR and contributes to AML1/ETO''s ability to repress proliferation and differentiation of primary bone marrow cells as well as to enhance their self renewal in vitro. We solved the solution structure of the MYND domain and show it to be structurally homologous to the PHD and RING finger families of proteins. We also determined the solution structure of an MYND-SMRT peptide complex. We demonstrated that a single amino acid substitution that disrupts the interaction between the MYND domain and the SMRT peptide attenuated AML1/ETO''s effects on proliferation, differentiation, and gene expression. [Copyright &y& Elsevier]

Summary: AML1/ETO is the chimeric protein resulting from the t(8;21) in acute myeloid leukemia. The Nervy homology 2 (NHR2) domain in ETO mediates oligomerization and AML1/ETO''s interactions with ETO, MTGR1, and MTG16, and with the corepressor molecules mSin3A and HDAC1 and HDAC3. We solved the NHR2 domain structure and found it to be an α-helical tetramer. We show that oligomerization contributes to AML1/ETO''s inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and affects AML1/ETO''s activity on several endogenous genes. Oligomerization is also required for AML1/ETO''s interactions with ETO, MTGR1, and MTG16, but not with other corepressor molecules. [Copyright &y& Elsevier]

Several substituted 4-aryloxy- and 4-arylsulfanyl-phenyl-2-aminothiazoles were synthesized and evaluated for cytotoxic activity against estrogen-positive, estrogen-negative, and adriamycin-resistant human breast cancer cell lines. 4-[4′-(3,4-Dichlorophenoxy)-phenyl]-thiazol-2-yl ammonium iodide demonstrated potent activity against both estrogen-positive and negative breast cancer cell lines with low micromolar (μM) GI50 values. In addition, we have identified several 2-aminothiazoles that demonstrated selective potency for the adriamycin-resistant and estrogen-negative breast cancer cell lines. The results suggest that these 2-aminothiazoles represent lead compounds for evaluation in animal models of breast cancer. [Copyright &y& Elsevier]

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways and in human disease. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancers. CBFs consist of a DNA-binding CBFα subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBFβ subunit. CBFα binds DNA in a sequence-specific manner, whereas CBFβ enhances DNA binding by CBFα. Both DNA binding and heterodimerization with CBFβ are mediated by a single domain in the CBFα subunits known as the "Runt domain." We analyzed the energetic contribution of amino acids in the Runx1 Runt domain to heterodimerization with CBFβ. We identified two energetic "hot spots" that were also found in a similar analysis of CBFβ (Tang, Y.-Y., Shi, J., Zhang, L., Davis, A., Bravo, J., Warren, A. J., Speck, N. A., and Bushweller, J. H. (2000) J. Biol. Chem. 275, 39579-39588). The importance of the hot spot residues for Runx1 function was demonstrated in in vivo transient transfection assays. These data refine the structural analyses and further our understanding of the Runx1-CBFβ interface. [ABSTRACT FROM AUTHOR]

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in hematopoiesis and in the development of bone, stomach epithelium, and proprioceptive neurons. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancer. CBFs consist of a DNA-binding CBFα subunit and a non-DNA-binding CBFβ subunit. DNA binding and heterodimerization with CBFβ are mediated by the Runt domain in CBFα. Here we report an alanine-scanning mutagenesis study of the Runt domain that targeted amino acids identified by structural studies to reside at the DNA or CBFβ interface, as well as amino acids mutated in human disease. We determined the energy contributed by each of the DNA-contacting residues in the Runt domain to DNA binding both in the absence and presence of CBFβ. We propose mechanisms by which mutations in the Runt domain found in hematopoietic and bone disorders affect its affinity for DNA. [ABSTRACT FROM AUTHOR]