Accessing Three-Dimensional Crystals with Incorporated Guests through Metal-Directed Coiled-Coil Peptide Assembly

Obtaining three-dimensional (3D) protein and peptide crystals on demand requires a precisely orchestrated hierarchical assembly of biopolymer building blocks. In this work, we disclose a metal-ion-mediated strategy to assemble trimeric coiled-coil peptides in a head-to-tail fashion into linear strands with interstrand interactions. This design led to hexagonal 3D peptide crystal formation within 30 min in the presence of divalent metal ions. The crystal morphology could be controlled by varying the metal ion/peptide ratio, resulting in hexagonal discs to rods. Diffraction studies elucidated the head-to-tail arrangement of the coiled-coil linear strands and their hexagonal, antiparallel packing within the crystal. Unsatisfied ligands at the hexagonal ends of the crystals were harnessed as a powerful means to direct His-tagged fluorophores to distinct locations within the crystals. Overall, the designed hierarchical assembly provides a facile means to obtain 3D peptide crystals and incorporate His-tag-based cargoes and may have potential use in drug delivery and sensor design

Systematic Analysis of the Physiological Importance of Deubiquitinating Enzymes

<div><p>Deubiquitinating enzymes (DUBs) are proteases that control the post-translational modification of proteins by ubiquitin and in turn regulate diverse cellular pathways. Despite a growing understanding of DUB biology at the structural and molecular level, little is known about the physiological importance of most DUBs. Here, we systematically identify DUBs encoded by the genome of <em>Drosophila melanogaster</em> and examine their physiological importance <em>in vivo</em>. Through domain analyses we uncovered 41 <em>Drosophila</em> DUBs, most of which have human orthologues. Systematic knockdown of the vast majority of DUBs throughout the fly or in specific cell types had dramatic consequences for <em>Drosophila</em> development, adult motility or longevity. Specific DUB subclasses proved to be particularly necessary during development, while others were important in adults. Several DUBs were indispensable in neurons or glial cells during developmental stages; knockdown of others perturbed the homeostasis of ubiquitinated proteins in adult flies, or had adverse effects on wing positioning as a result of neuronal requirements. We demonstrate the physiological significance of the DUB family of enzymes in intact animals, find that there is little functional redundancy among members of this family of proteases, and provide insight for future investigations to understand DUB biology at the molecular, cellular and organismal levels.</p> </div

Differences in distribution and levels of ubiquitinated species in whole fly lysates.

<p>Shown are western blots of whole, newly-eclosed adult flies homogenized in SDS lysis buffer and electrophoresed in SDS-PAGE gels. Experimental groups were heterozygous for sqh-Gal4 and UAS-RNAi. Controls were heterozygous for sqh-Gal4 on the isogenic background of RNAi lines. Boxes highlight some areas with visible differences in ubiquitinated species. Western blots are representative of at least three independent repeats with similar results. Underlined: ubiquitous knockdown led to phenotype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043112#pone-0043112-g003" target="_blank">Figure 3</a>).</p

Phenotypic distribution by developmental stage.

<p>Histograms show the number of DUBs whose knockdown led to phenotype at each stage. A) sqh-Gal4 (ubiquitous) driver; details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043112#pone-0043112-g003" target="_blank">Figure 3</a>. B) strong elav-Gal4 (pan-neuronal) driver; details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043112#pone-0043112-g005" target="_blank">Figure 5</a>. In cases where two different RNAi lines targeting the same DUB led to phenotype in different stages, only the earlier stage was counted. Instances where knockdown of a specific DUB by one RNAi line led to defects in two stages were counted for each stage (e.g. knockdown of CG3416 led to death in both larval and pupal stages).</p

Gene dosage effects with pan-neuronal knockdown.

<p>List of phenotypes observed when individual fly DUBs were knocked down by pan-neuronal drivers with different expression strengths.</p

List of phenotypes when individual DUBs are knocked down throughout the fly.

<p>Listed are the strongest phenotypes associated with ubiquitous knockdown of individual fly DUBs. Diamonds highlight DUBs whose knockdown led to observable phenotypes. Experimental groups consisted of flies heterozygous for sqh-Gal4 and UAS-RNAi. Controls were heterozygous for sqh-Gal4 on the isogenic background of RNAi lines. “Dead soon after eclosion” category denotes flies that eclosed successfully from the pupal case but fell on food and died within a few hours. Late motility phenotype means it was first observed 20 or more days after eclosing from the pupal case.</p

Gal4 drivers.

<p>Flies carrying UAS-GFP were crossed to flies carrying the Gal4 driver specified in the figure. Newly-eclosed adults heterozygous for UAS-GFP and the Gal4 driver were homogenized in SDS lysis buffer and loaded into SDS/PAGE gels. Western blots show how strongly each Gal4 driver expresses UAS-GFP. Blots were probed with anti-GFP and anti-tubulin antibodies. Ubiquitous driver: sqh-Gal4. Pan-neuronal drivers: elav-Gal4.</p

Comprehensive list of fly DUBs and their physiological significance.

<p>Listed are all the fly DUBs that we identified, highlighting previously reported functions and our current findings. Not tested: DUBs that we did not examine either because of lack of reagents, too many non-specific targets from existing RNAi lines, or because the function of these DUBs is well characterized in flies. Empty cells: as of this publication, no information had been previously reported for these DUBs.</p

Stabilization of an Unusual Salt Bridge in Ubiquitin by the Extra C‑Terminal Domain of the Proteasome-Associated Deubiquitinase UCH37 as a Mechanism of Its Exo Specificity

Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs); ∼100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood. UCH37, a cysteine DUB conserved from fungi to humans, is a proteasome-associated factor that regulates the proteasome by sequentially cleaving polyubiquitin chains from their distal ends, i.e., by exo-specific deubiquitination. In addition to the catalytic domain, the DUB features a functionally uncharacterized UCH37-like domain (ULD), presumed to keep the enzyme in an inhibited state in its proteasome-free form. Herein we report the crystal structure of two constructs of UCH37 from <i>Trichinella spiralis</i> in complex with a ubiquitin-based suicide inhibitor, ubiquitin vinyl methyl ester (UbVME). These structures show that the ULD makes direct contact with ubiquitin stabilizing a highly unusual intramolecular salt bridge between Lys48 and Glu51 of ubiquitin, an interaction that would be favored only with the distal ubiquitin but not with the internal ones in a Lys48-linked polyubiquitin chain. An inspection of 39 DUB–ubiquitin structures in the Protein Data Bank reveals the uniqueness of the salt bridge in ubiquitin bound to UCH37, an interaction that disappears when the ULD is deleted, as revealed in the structure of the catalytic domain alone bound to UbVME. The structural data are consistent with previously reported mutational data on the mammalian enzyme, which, together with the fact that the ULD residues that bind to ubiquitin are conserved, points to a similar mechanism behind the exo specificity of the human enzyme. To the best of our knowledge, these data provide the only structural example so far of how the exo specificity of a DUB can be determined by its noncatalytic domain. Thus, our data show that, contrary to its proposed inhibitory role, the ULD actually contributes to substrate recognition and could be a major determinant of the proteasome-associated function of UCH37. Moreover, our structures show that the unproductively oriented catalytic cysteine in the free enzyme is aligned correctly when ubiquitin binds, suggesting a mechanism for ubiquitin selectivity