Functional and structural consequences of aromatic residue substitutions within the kringle-2 domain of tissue-type plasminogen activator.
Aromatic amino acid residues within kringle domains play important roles in the structural stability and ligand-binding properties of these protein modules. In previous investigations, it has been demonstrated that the rigidly conserved Trp25 is primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2tpA), whereas Trp63, Trp74, and Tyr76 function in omega-amino acid ligand binding, and, to varying extents, in stabilizing the native folding of this kringle module. In the current study, the remaining aromatic residues of K2tPA, viz., Tyr2, Phe3, Tyr9, Tyr35, Tyr52, have been subjected to structure-function analysis via site-directed mutagenesis studies. Ligand binding was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr35 destabilized the interaction of the ligand with the variant kringle. In addition, as reflected in the values of the melting temperatures, changes at Tyr9 and Tyr52 generally destabilized the native structure of K2tPA to a greater extent than changes at Tyr2, Phe3, and Tyr35. Taken together, results to date show that, in concert with predictions from the crystal structure of K2tpA, ligand binding appears to rely most on the integrity of Trp63 and Trp74, and aromaticity at Tyr76. With regard to aromatic amino acids, kringle folding is most dependent on Tyr9, Trp25, Tyr52, Trp63, and Tyr76. As yet, no obvious major roles have been uncovered for Tyr2, Phe3, or Tyr35 in K2tpA.
Chang
Y
Y
Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA.
Nilsen
S L
SL
Castellino
F J
FJ
D019943:Amino Acid Substitution; D002152:Calorimetry, Differential Scanning; D005453:Fluorescence; D018082:Kringles / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D010649:Phenylalanine; D010959:Tissue Plasminogen Activator