title: Structural determinants in protein folding: a single conserved hydrophobic residue determines folding of EGF domains.
abstract: The epidermal growth factor (EGF) domain is evolutionarily conserved despite hypervariability in amino acid sequences. They fold into a three-looped conformation with a disulfide pairing of C(1)-C(3), C(2)-C(4) ,and C(5)-C(6). To elucidate the structural determinants that dictate the EGF fold, we selected the fourth and fifth EGF domains of thrombomodulin (TM) as models; the former domain folds into the canonical conformation, while the latter domain folds with alternate disulfide pairing of C(1)-C(2), C(3)-C(4), and C(5)-C(6). Since their third disulfide (C(5)-C(6)) is conserved, we examined the folding tendencies of synthetic peptides corresponding to truncated domain four (t-TMEGF4) and five (t-TMEGF5), encompassing the segment C(1) to C(4). These peptides fold into their respective disulfide isoforms indicating that they contain all the required structural determinants. On the basis of the folding tendencies of these peptides in the absence and presence of 6 M Gn·HCl or 0.5 M NaCl, we determined that hydrophobic interactions are needed for the canonical EGF fold but not for the noncanonical fold. Sequence alignment of extant EGF domains and examination of their three-dimensional structures allowed us to identify a highly conserved hydrophobic residue in intercysteine loop 3 as the key contributor, which nucleates the hydrophobic core and acts as the lynch pin. When this hydrophobic residue (Tyr25) was substituted with a more hydrophilic Thr, the hydrophobic interactions were disrupted, and t-TMEGF4-Y25T folds similar to t-TMEGF5. Taken together, our results for the first time demonstrate that a single conserved hydrophobic residue acts as the key determinant in the folding of EGF domains.
authors.affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543, Singapore.
authors.forename: A S Angie
authors.initials: AS
authors.lastname: Ng
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004815:Epidermal Growth Factor / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms; D016415:Sequence Alignment
chemical_list: D020033:Protein Isoforms; D004815:Epidermal Growth Factor