Cap hydrolysis by Dcp2 is a critical step in several eukaryotic mRNA decay pathways. Processing requires access to cap-proximal nucleotides and the coordinated assembly of a decapping mRNP, but the mechanism of substrate recognition and regulation by protein interactions have remained elusive. Using NMR spectroscopy and kinetic analyses, we show that yeast Dcp2 resolves interactions with the cap and RNA body using a bipartite surface that forms a channel intersecting the catalytic and regulatory Dcp1-binding domains. The interaction with cap is weak but specific and requires binding of the RNA body to a dynamic interface. The catalytic step is stimulated by Dcp1 and its interaction domain, likely through a substrate-induced conformational change. Thus, activation of the decapping mRNP is restricted by access to 5'-proximal nucleotides, a feature that could act as a checkpoint in mRNA metabolism.
Human CA150, a transcriptional activator, binds to and is co-deposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded β-sheet that folds
in vitro
in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue nonnative β-hairpin. The structure we report has similarities to the hairpin formed by the A
β
(1–40)
protofilament, yet also contains closely packed side-chains in a “steric zipper” arrangement found in the cross-β spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.
Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding. An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity. Phosphoactivation of DUBA represents an unprecedented mode of protease regulation and a clear link between two major cellular signal transduction systems: phosphorylation and ubiquitin modification.
In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein). Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations. Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment is presented, using spectra of the chicken alpha-spectrin SH3 domain (62 residues), alphaB-crystallin (175 residues) and outer membrane protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Calpha, Cbeta, C' and N resonances in the core domain of alphaB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini of the protein).
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