Polyalanine

We solvated terminally capped alanine-based helices with the sequence Ace-(Ala)₄Xaa(Ala)₄-NMA, where Xaa is any 1 of the 20 naturally occurring amino acids other than Gly, Ala, and Pro, in a cubic box with sides of ∼27Å containing ∼600 water molecules. Protonation states were chosen to correspond to neutral pH. Because the goal was to compare the rotamer distributions observed in MD simulations of these peptides to the distributions observed in helices, we applied a weak restraint to both the φ and ϕ torsion angles to ensure that the peptides stayed helical. These restraints were of the form:

with reference values (θ₀) of 122° and 133° for φ and ϕ, respectively, and a force constant (k_θ) of 1 kcal mol⁻¹. We note that although the reference values do not correspond to the helical region of the Ramachandran map, this cosine series acts as a restraint that ensures that the peptide remains in a helical conformation throughout the entire simulation without noticeably influencing the side-chain motion.
Each system was equilibrated at 300 K and 1 atm with 2.4 ns of MD simulation in the NPT ensemble. Then, MD simulations were carried out in the NVT ensemble for 720 ns using the Nosé-Hoover thermostat with a relaxation time of 1 ps. All simulations were performed using the Desmond MD program16 version 2.1.1.0 and either the Amber ff99SB7 (link) or the modified Amber ff99SB force field described herein, which we have termed ff99SB-ILDN. All bonds involving hydrogen atoms were constrained with the SHAKE algorithm.17 (link) A cutoff of 10 Å was used for the Lennard-Jones interaction and the short-range electrostatic interactions. The smooth particle mesh Ewald method18 with a 32 × 32 × 32 grid and a fourth-order interpolation scheme was used to compute the long-range electrostatic interactions. The pairlists were updated every 10 fs with a cutoff of 10.5 Å. We used a multistep RESPA scheme19 for the integration of the equations of motion with timesteps of 2.0, 2.0, and 6.0 fs for the bonded, short-range nonbonded, and long-range nonbonded interactions, respectively. To check for potential biases introduced by long-range interactions between peptides in periodic images, we repeated these simulations for four of the amino acids (Xaa: Ile, Leu, Asp, and Asn) using a larger box with side length 37 Å. We found that the results of these control simulations were within error of those using the smaller box sizes.

Microscale thermophoresis (MST) analyses of the SCCH domain:polyalanine peptide interaction were carried out using our Monolith NT.115 (Nano Temper Technologies). A peptide containing a tandem of 7 alanine residues was labeled with Cy5 (GL Biochem Shanghai Ltd.), and was used at constant final concentration of 100 nM. The labeled peptide was mixed with the ligand, a purified recombinant unlabeled UBA6 (623-889) or UBA1 (624-891) SCCH domains. 1:2 serial dilutions of the ligands from 200 µM to 1.56 µM final concentration were assayed. After a short incubation, the samples were loaded into premium glass capillaries before data collection. All MSTs were performed twice at Excitation Power 30% of the LED and MST power of 20% and 40%.

Cells were lysed on ice in immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 2 mM MgCl₂, 0.5% NP-40), supplemented with a protease inhibitors cocktail before use. In IP experiments performed in separated cell fractions, cells were lysed in IP buffer containing 0.1% NP40. Supernatant was kept as a cytoplasmic fraction and the nuclear pellet was dispersed with IP buffer and passed through 25 and 27 gauge needle then sonicated to ensure extraction of nuclear proteins. For polyubiquitination experiments, cells were treated with a proteasome inhibitor MG132 (10 μM) during the last 6 h before lysis with the IP buffer supplemented with 1 mM PMSF and 10 mM iodoacetamide.
Whole-cell lysates obtained by centrifugation were incubated with 2–5 μg of antibody overnight at 4 °C, followed by 2 h incubation with Protein A-Sepharose CL-4B (Cytiva, 17-0780-01). The immunocomplexes were then washed three times with IP buffer, and boiled at 95 °C for 5 min in Laemmli sample buffer containing 5% beta-mercaptoethanol, before being separated by SDS–PAGE for western blotting assays. For experiments analyzing endogenous UBA6-USE1 interaction in USE1 ΔPolyAla KO cells, a cross-linking step was performed with formaldehyde prior to cell lysis and IP. For experiments to examine the binding of isolated polyalanine stretches, a pre-clearing step was performed by incubating the whole cell lysates with 25 µl beads for 2 h at 4 °C. The beads were then discarded, and the cell lysates were incubated with antibody overnight as already described. For experiments analyzing ubiquitin load on USE1 under expression of polyalanine disease proteins, the different polyalanine constructs were expressed in HEK293T cells for 72 h while the FLAG-USE1 was expressed in the cells for the last 24 h.

USE1/UBE2Z human homologs were searched against the Uniprot (Pubmed id 29425356) and NCBI databases using BLAST (Pubmed id 2231712). Prosite (Pubmed id 23161676) was used to scan for alanine residues motifs with between 6 and 10 continuous alanine residues in the BLAST results (a search for proteins containing polyalanine stretches in the ubiquitin cascades). Alignments of the E2 family in vertebrates and across all databases were calculated using MAFFT (Pubmed id 28968734). The figures were generated using Jalview (Pubmed id 19151095).