Homology model generation and protein-protein docking

The I-TASSER server (74) was used to create homology models of PER2(683–872) wild-type and mutant variants. Sequences were uploaded to the server in FASTA format. There were no restraints guiding modeling, homologous templates were not excluded, and secondary structures for specific residues were unbiased. The server uses templates from the Protein Data Bank (PDB) database to predict secondary structure of the query protein using LOMETS 4 (local meta-threading-server). Alternatively, the server uses ab initio modeling to assign secondary structures. Clustering was then performed to find the lowest free-energy model using SPICKER (75). The model with the highest C score value was then energy minimized using the Molecular Operating Environment with Amber12EHT parameters and subsequently assessed for the quality of the model using online servers including SWISS-MODEL (76, 77), ProSA (78), and VERIFY3D (79, 80). Models of all three constructs showed favorable energies relevant to their ANOLEA, PROCHECK, and z scores, as well as favorable three-dimensional structure and side-chain placements, and were deemed acceptable (fig. S5). Here, the three models were validated and used in confidence in further protein-ubiquitin docking experiments.

Protein binding interfaces were predicted by docking between ubiquitin [PDB: 1UBQ (81)] and each model of the PER2 fragments. The Schrödinger software suite (2017.2) and the BioLuminate interface, which uses the PIPER docking module, were used for interface determination. No biased or interfaced residue was set at the onset of docking. All PER2 models were treated equally in regard to how and where ubiquitin molecules were predicted to interact. Thirty structures for each PER2:ubiquitin docking pair were obtained and clustered using pairwise root mean square deviation, and key residues located at the interface were identified. Data files are available from the Virginia Tech Institutional Data Repository, VTechData, doi:10.7294/W4JW8C2R.