A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

Open access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesCASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions. Results We demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size. Conclusions This study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.Ye

Crystal Structure of USP7 Ubiquitin-like Domains with an ICP0 Peptide Reveals a Novel Mechanism Used by Viral and Cellular Proteins to Target USP7

<div><p>Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.</p></div

The crystal structure of the Ubl123-ICP0 peptide complex.

<p>(A) Interaction between Ubl123 and ICP0 peptide. Ubl123 is shown in cartoon representation and the ICP0 peptide in stick representation. (B) Electrostatic surface representation with acidic regions shown in red and basic regions in blue. (C) Front view of the ICP0 binding site. Interactions between Ub123 and ICP0 are highlighted by dashed lines. (D) Surface representation of the binding pocket with stick model of ICP0. (E) Side view of the ICP0 binding site. Interactions between Ub123 and ICP0 are highlighted by dashed lines.</p

Two conformational states of Ubl123.

<p>(A) Extended conformation as observed in Se-Ubl123 (this work) and native USP7-CTD (PDB ID 2YLM). (B) Compact conformation observed for native Ubl123 (this work). (C) Details of contact formation between Ubl1 and Ubl3. (D) Details of contact formation between Ubl1 and Ubl3. (E) Details of the Ubl2-Ubl3 hinge. In F, the extended conformation is partially given for direct comparison; note that S814 is now occupying the former position of the backbone nitrogen atom of N815. A pink arrow indicates the shift from the extended to the compact conformation. The entire Ubl3 domain appears to rotate against Ubl12, the pivot point being between residue H792 and R793. Dashed lines indicate hydrogen bonds and salt-bridges, distances are given in Å. (F) Details of the Ubl2-Ubl3 hinge. In F, the extended conformation is partially given for direct comparison; note that S814 is now occupying the former position of the backbone nitrogen atom of N815. A pink arrow indicates the shift from the extended to the compact conformation. The entire Ubl3 domain appears to rotate against Ubl12, the pivot point being between residue H792 and R793. Dashed lines indicate hydrogen bonds and salt-bridges, distances are given in Å.</p