Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation.
Background: Archaeoglobus fulgidus ferritin (AfFtn) assembles with unique tetrahedral symmetry and four large pores. Results: The AfFtn K150A/R151A double mutant forms a closed octahedral assembly with reduced iron release rates relative to the tetrahedral assembly. Conclusion: The K150A/R151A substitution alters the symmetry type of the ferritin cage. Significance: The AfFtn can be modulated for tuning molecular release from the cavity.
Both cardiac and skeletal calsequestrin (CASQ2 and CASQ1) serve as a major Ca(2+) storage/buffer protein in the sarcoplasmic reticulum (SR) by sequestering and releasing large numbers of Ca(2+) ions during each muscular contraction and relaxation cycle. CASQ isolated from various species often exists in a phosphorylated form, but phosphorylation's role is not yet understood. Here, the authors identified two phosphorylation sites, Ser(385) and Ser(393), for the first time, in human CASQ2 (hCASQ2) by mass-spectroscopy and evaluated the consequences of such phosphorylation. Substitution of these two serines with phosphoserine-mimicking aspartic-acid residues results in a significant increase in helical content, solubility and Ca(2+)-binding capacity above 6 mM [Ca(2+)]. However, neither substitution of Ser(385) nor Ser(393) alone produce any significant changes. Based on the crystal structures of hCASQ2, Ca(2+) binding capacity data, turbidity, and light scattering profiles, it was propose that phosphorylation at these two positions produces a disorder-to-order or coil-to-helix transition of the C-terminus, which in turn provides a more stable network of polyanions. Therefore, considering all the previous reports and the new data, the observed dynamic in vivo phosphorylation of CASQ could provide the basis not only for effective regulation of Ca(2+) buffering capacity, but also for the junctional SR trafficking mechanism.
Human UDP-α-d-xylose synthase (hUXS) is a member of the extended short chain dehydrogenase/reductase (SDR) family of enzymes. Previous crystallographic studies have shown that hUXS conserves the same dimeric quaternary structure observed in other SDR enzymes. Here, we present evidence that hUXS also forms a tetramer in solution that is important for activity. Sedimentation velocity studies show that two hUXS dimers undergo a concentration-dependent association to form a tetramer with a Kd of 2.9 μM. The tetrameric complex is also observed in small-angle X-ray scattering (SAXS). The specific activity for the production of the reaction intermediate UDP-α-d-4-keto-xylose displays a hyperbolic dependence on protein concentration that is well modeled by an isotherm using the 2.9 μM Kd of the tetramer. Likewise, the rate of UDP-α-d-xylose production in the presence of increasing concentrations of the small molecule crowder trimethylamine N-oxide is consistent with the formation of a higher activity tetramer. We present several possible structural models of the hUXS tetramer based on (i) hUXS crystal packing, (ii) homology modeling, or (iii) ab initio simulated annealing of dimers. We analyze the models in terms of packing quality and agreement with SAXS data. The higher activity of the tetramer coupled with the relative instability of the complex suggests that an association-dissociation mechanism may regulate hUXS activity.
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