Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at 
conditions of glucose deprivation. Pck1 is a rate-limiting gluconeogenic enzyme, 
where its deficiency or mutation contributes to serious clinical situations as 
neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a 
target for proteasomal degradation through its proline residue at the 
penultimate position, recognized by Gid4 E3 ligase, but with a lack of 
informative structural details. In this study, we delineate the localized 
sequence motif, degron, that specifically interact with Gid4 ligase and unravel 
the binding mode of Pck1 to the Gid4 ligase by using molecular docking and 
molecular dynamics. The peptide/protein docking HPEPDOCK web server along with 
molecular dynamic simulations are applied to demonstrate the binding mode and 
interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently 
solved structure of Gid4 ligase. Results unveil a distinct binding mode of the 
mutated peptide compared with the wild type despite having comparable binding 
affinities to Gid4. Moreover, the four-residue peptide is found insufficient for 
Gid4 binding, while the seven-residue peptide suffices for binding to Gid4. The 
amino acids S134, K135, and N137 in the loop L1 (between β1 and β2) of the Gid4 
are essential for the stabilization of the seven-residue peptide in the binding 
site of the ligase. The presence of Val4 instead of Lys4 smashes the H-bonds 
that are formed between Lys4 and Gid4 in the wild type peptide, making the 
peptide prone to bind with the other side of the binding pocket (L4 loop of 
Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant 
Pck1 peptide is introduced. This opens the door to explore the mutation effects 
on the binding mode and smooth the path to target protein degradation by design 
competitive and non-competitive inhibitors.