2.1. Structure prediction of auto-inhibitory subdomain through threading

Determining the structure of the AI domain is the cornerstone to understanding the molecular organisation, autoinhibitory mechanism and activation of the full length PKGIα. In PKGIα, there is no experimentally solved structure for the region spanning amino acids 47 to 102. This sequence region covering 25 functionally important amino acids of the AI domain binds and inactivates the catalytic region through the pseudo-substrate motif, to downregulate the activity of PKGIα. To define the structure of the AI domain, the amino acid sequence of the query structure was retrieved from UniProt (Uniprot ID: {"type":"entrez-protein","attrs":{"text":"Q13976","term_id":"6225588","term_text":"Q13976"}}Q13976) [21] and a PSI-BLAST search was performed against the Protein Data Bank (PDB) templates [22], [23]. All the templates identified in the BLAST search were < 20% in sequence identity to the query sequence. In the absence of an appropriate template, a threading technique was employed for 3D structure prediction. The AI domain accounts for 25 residues which was too short for the structure prediction on I-TASSER suite [24] hence, the sequence length was extended to 55 amino acids. This was done to reduce false positivity in the results and achieve a good quality model. I-TASSER identified the 10 best templates by using LOMETs and generated multiple template sequence alignments using a number of threading programs. The initial model was selected based on TMalign [25] and C-score [26]. Model refinement was performed in UCSF Chimera [27] and evaluation of the refined model was achieved using RAMPAGE and MolProbity [28].