Structural Determinants in the folding of epidermal growth factor (EGF)-Like domains

The epidermal growth factor (EGF)-like domain is an evolutionarily conserved modular protein subunit. Despite hypervariability of amino acid sequences in their inter-cysteine region, they preferentially fold into a three-looped conformation with a disulfide pairing of C₁-C₃, C₂-C₄, C₅-C₆. To elucidate the structural determinants that dictates the canonical EGF-like domain fold, we had chosen the fourth and fifth EGF-like domain of thrombomodulin (TM) as models. While the fourth EGF-like domain folds into the canonical conformation, the fifth EGF-like domain does not and possesses an alternate disulfide pairing of C₁-C₂, C₃-C₄, C₅-C₆. We examined the folding tendencies of two synthetic peptides corresponding to truncated versions of TM EGF-like domain four and five under air oxidation and redox folding conditions. By identifying the structural isoforms obtained in the folding reaction using regiospecifically-synthesized conformers as controls, we determined that the last segment of both domains (encompassing C₅ and C₆) do not influence the tendencies to fold into their respective native conformations. When folded under denaturing conditions, the folding tendency of the fourth EGF-like domain changes to that of the C₁-C₂, C₃-C₄ conformer. Conversely, the addition of denaturant did not affect the folding tendency of the fifth EGF-like domain. This suggests that side chain interactions are crucial for achieving the canonical EGF-like domain fold but not for the non-canonical fold. Folding under high salt content did not disrupt the folding tendencies of both domains and result in slight increase of the C₁-C₃, C₂-C₄ conformer in both cases. This suggests that hydrophobic interaction, but not electrostatic interaction, is the key in the achieving the canonical fold of EGF-like domains.

Author dc:creator

• NG AH SOCK ANGIE

• protein folding code, structural determinants, epidermal growth factor-like domains