Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency

Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABA_ARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABA_ARs and GlyRs together control postsynaptic receptor clustering

Specificity of the E1-E2-E3 Enzymatic Cascade for Ubiquitin C‑Terminal Sequences Identified by Phage Display

Ubiquitin (UB) is a protein modifier that regulates many essential cellular processes. To initiate protein modification by UB, the E1 enzyme activates the C-terminal carboxylate of UB to launch its transfer through the E1-E2-E3 cascade onto target proteins. In this study, we used phage display to profile the specificity of the two human E1 enzymes, Ube1 and Uba6, toward the C-terminal sequence of UB ending with ⁷¹LRLRGG⁷⁶. Phage selection revealed that while Arg72 of UB is absolutely required for E1 recognition, UB residues at positions 71, 73, and 74 can be replaced with bulky aromatic side chains, and Gly75 of UB can be changed to Ser, Asp, and Asn for efficient E1 activation. We have thus found that the E1 enzymes have substantial promiscuity regarding the UB C-terminal sequence. The UB variants from phage selection can also be transferred from E1 to E2 enzymes; however, they are blocked from further transfer to the E3 enzymes. This suggests that the C-terminal sequence of UB is important for its discharge from E2 and subsequent transfer to E3. In addition, we observed that the Leu73Phe and Leu73Tyr single mutants of UB are resistant to cleavage by deubiquitinating enzymes (DUBs), although they can be assembled by the E1-E2-E3 cascade into poly-UB chains, thus indicating differences in UB C-terminal specificities between the E1 and DUBs. Consequently these UB mutants may provide stability to UB polymers attached to cellular proteins and facilitate the elucidation of the biological signals encoded in the UB chains

Inhibiting the Protein Ubiquitination Cascade by Ubiquitin-Mimicking Short Peptides

Short heptapeptides were identified to function as ubiquitin (UB) mimics that are activated by E1 and form thioester conjugates with E1, E2, and HECT type E3 enzymes. The activities (<i>k</i>_cat/<i>K</i>_1/2) of E1 with the UB-mimicking peptides are 130–1,400-fold higher than the equally long peptide with the native C-terminal sequence of UB. By forming covalent conjugates with E1, E2, and E3 enzymes, the UB-mimicking peptides can block the transfer of native UB through the cascade

The Nedd8 transfer cascade and phage selection of UB variants for NAE activation.

<p>(a) Nedd8 is first activated by heterodimeric NAE composed of the APPBP1 and Uba3 subunits to form a Nedd8∼NAE thioester conjugate followed by the transfer of Nedd8 to the E2 enzyme Ubc12. The Nedd8∼Ubc12 conjugate is then bound to the cullin-RING complex for cullin modification by Nedd8. (b) For phage selection of UB variants reactive with NAE, a PCP-NAE fusion was labeled with biotin and immobilized on a streptavidin plate. Phage library displaying UB with randomized C-terminal sequences was added to the streptavidin plate with ATP to allow UB variants to form thioester conjugates with NAE. Phage displaying NAE reactive UB variants were eluted by cleaving the thioester bond in the UB∼NAE conjugate by DTT.</p

The activities of UB variants and the corresponding C-terminal peptides with NAE measured by the ATP-PP_i exchange assay.

<p>(a) Activities of full-length Nedd8 or UB variants with NAE. (B) Activities of the heptameric Nedd8-mimicking peptides (sequences indicated in single letter code) derived from the UB variants with NAE.</p

Alignment of the C-terminal sequences of the phage selected UB clones which are reactive with NAE.

<p>Sequences are grouped according to the identities of the residues selected at position 72. UB residues 71–75 were randomized in the library and they are designated with the red stars. Names of UB variants that were expressed and further analyzed for reactivity with NAE are shown in red. Numbers of times selected refers to how often a UB clone was identified among the 58 clones sequenced after the fifth round of phage selection.</p

Measuring the activity of biotin-labeled PCP-NAE fusion protein immobilized on the streptavidin plate by ELISA.

<p>(a) Assaying the formation of Nedd8∼NAE thioester conjugate immobilized to the streptavidin plate by a mouse anti-HA antibody and an anti-mouse antibody conjugated to horse radish peroxidase (HRP). (b) Results of the ELISA assay with 10× dilution of HA-Nedd8 across the plate. 5 µM Nedd8 was added to the wells in the first column of the streptavidin plate. In the control reactions, either PCP-NAE was not immobilized to the plate or ATP was not added to the reaction.</p

Transfer of Nedd8-mimicking peptides to NAE, Ubc12 and cullin-3, and inhibition of Nedd8 transfer through the cascade enzymes.

<p>(a) Transfer of peptides pN1, pN7 and pN26 to NAE, Ubc12 and cullin 3. The peptides were conjugated to biotin at their N-termini. The Uba3 subunit of NAE formed the thioester conjugate with the peptides. The Western blot was probed with streptavidin conjugated to HRP. (b) Inhibiting the formation of Nedd8∼NAE conjugates by the Nedd8-mimicking peptides. (c) Inhibiting Nedd8 transfer to Ubc12 and cullin-3 proteins by the peptides.</p

Transfer of phage selected UB variants to NAE (E1), Ubc12 (E2), and cullin-3.

<p>HA-tagged wt UB, wt Nedd8 and UB variants were used in the assay. In the control reaction, either Ubc12 or cullin-3 in complex with Rbx1 was missing. The blots were probed with a mouse anti-HA antibody and a goat anti-mouse IgG antibody linked to HRP.</p

Enrichment of UB variants reactive with NAE over iterative rounds of phage selection.

<p>Enrichment of UB variants reactive with NAE over iterative rounds of phage selection.</p