The Survival Motor Neuron (SMN) protein forms the oligomeric core of a multi-protein complex that functions in spliceosomal snRNP biogenesis. Loss of function mutations in the SMN gene cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Nearly half of the known SMA patient missense mutations map to the SMN YG-box, a highly conserved oligomerization domain of unknown structure that contains a (YxxG)3 motif. Here we report that the SMN YG-box forms helical oligomers similar to the glycine zippers found in transmembrane channel proteins. A novel network of tyrosine-glycine packing between helices drives formation of soluble YG-box oligomers, providing a structural basis for understanding SMN oligomerization and for relating defects in oligomerization to the mutations found in SMA patients. These results have important implications for advancing our understanding of SMN function and glycine zipper-mediated helix-helix interactions.

Background:The survival motor neuron (SMN) protein forms oligomeric complexes involved in ribonucleoprotein (RNP) biogenesis. Results: SMN forms stable dimers, which in turn self-associate to form tetramers and octamers. Conclusion: SMN complexes form discrete oligomers with unusually large hydrodynamic sizes. Significance: Understanding the oligomeric nature of SMN provides an important foundation for exploring the biochemical bases of RNP assembly and spinal muscular atrophy.

We have developed a novel series of potent and selective factor Xa inhibitors that employ a key 7-fluoroindazolyl moiety. The 7-fluoro group on the indazole scaffold replaces the carbonyl group of an amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing 7-fluoroindazole 51a showed the 7-fluoro atom hydrogen-bonding with the N-H of Gly216 (2.9 A) in the peptide backbone. Thus, the 7-fluoroindazolyl moiety not only occupied the same space as the carbonyl group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with the protein's beta-sheet domain. The structure-activity relationship for this series was consistent with this finding, as the factor Xa inhibitory potencies were about 60-fold greater (DeltaDelta G approximately 2.4 kcal/mol) for the 7-fluoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis of these factor Xa inhibitors is also described.

The BTB/POZ domain defines a conserved region of about 120 residues and has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RAR␣ (retinoic acid receptor ␣) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. We have overexpressed, purified, characterized, and crystallized the BTB/POZ domain from PLZF (PLZF-BTB/POZ). Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a K d below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition with a T m of 70.4°C, and a ⌬G of 12.8 ؎ 0.4 kcal/ mol. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% ␤-sheet. We have prepared crystals of the PLZF-BTB/POZ that are suitable for a high resolution structure determination using x-ray crystallography. The crystals form in the space group I222 or I2 1 2 1 2 1 with a ‫؍‬ 38.8, b ‫؍‬ 77.7, and c ‫؍‬ 85.3 Å and contain 1 protein subunit per asymmetric unit with approximately 40% solvent. Our data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/ POZ domain function.The PLZF BTB/POZ domain, named for its presence in the Drosophila proteins Broad Complex, tramtrack, and bric a brac (BTB) (1) and its homology with several poxvirus proteins and zinc finger proteins (POZ) (2), is an evolutionarily conserved motif of about 120 residues (Fig. 1) found in an increasing number of proteins having a variety of functions (1-4). Proteins containing a BTB/POZ domain have been identified in poxvirus, Caenorhabditis elegans, Drosophila, and human and are generally found at the N terminus of either actin-binding or, more commonly, nuclear DNA-binding proteins (4). Proteins containing a BTB/POZ domain have been associated with diverse functions including nucleosome/chromosome disruption, pattern formation, metamorphosis, oogenesis, and eye and limb development (5-11).Biological relevance for the function of BTB/POZ domains has come from the study of the PLZF (for promyelocytic leukemia zinc finger) oncoprotein, associated with acute promyelocytic leukemia (APL).1 APL results from the malignant proliferation of cells blocked at the stage of promyelocytic differentiation and accounts for about 10% of all myeloid leukemias (...