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 mo dules. In previous investigations, it has been demonstrated that the rigidl y conserved Trp(25) is, primarily involved in stabilizing the conformation of the kringle-2 domain of tissue-type plasminogen activator (K2(tPA)), whe reas Trp(63), Trp(74) and Tyr(76) function in cu-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 K2(tPA), viz., Tyr(2), Phe(3), Tyr(9), Tyr(35), Tyr(52) have been subjected to struc ture-function analysis via site-directed mutagenesis studies. Ligand bindin g was not significantly influenced by conservative amino acid mutations at these residues, but a radical mutation at Tyr(35) destabilized the interact ion of the ligand with the variant kringle. In addition, as reflected in th e values of the melting temperatures, changes at Tyr(9) and Tyr(52) general ly destabilized the native structure of K2(tPA) to a greater extent than ch anges at Tyr(2), Phe3, and Tyr(35). Taken together, results to date show th at, in concert with predictions from the crystal structure of K2(tPA). liga nd binding appears to rely most on the integrity of Trp(63) and Trp(74), an d aromaticity at Tyr(76). With regard to aromatic amino acids, kringle fold ing is most dependent on Tyr(9), Trp(25), Tyr(52), Trp(63), and Tyr(76). AS yet, no obvious major roles have been uncovered for Tyr(2), Phe(3), or Tyr (35) in K2(tPA).