Metal ion—FTS nonapeptide interactions II. Copper(II) complex equilibria and structures under physiological conditions

Abstract

Zinc is essential to the physiological activity of thymulin, a metallohormone involved in T-cell differentiation. The association of Zn 2+ ions to the free nonapeptide (facteur thymique sérique, or FTS) plays a highly specific role in thymulin activity, even though other metal ions, such as Cu 2+, can partially restore it following incubation of the native hormone with Chelex 100. In order to throw light on the mechanisms through which metal ion—FTS association can influence thymulin activity, the present work reports an investigation into the stoichiometry and structure of copper(II) complexes with FTS under physiological conditions (37°C, 0.15 mol dm −3 NaCl). First, glass electrode potentiometry was used to study Cu(II)—FTS complex equilibria and to determine corresponding formation constants. Speciation calculations based on these constants were then used to design experiments with several spectroscopic techniques (electronic absorption, circular dichroism and EPR) which led to the determination of related structures. Except for the first bond to the Asn 9 C-terminal carboxylate group in the MLH species formed at low pH, copper coordination with FTS follows a rather unusual scheme. Classically, copper(II)-peptide bonding begins with the coordination of the Cu 2+ aqua ion to an amino or an imidazole nitrogen that serves as an anchor site to initiate metal chelation through successive deprotonation of peptide bonds as the pH is raised. In the present case, the ɛ-terminal amino group of the Lys 3 residue, which binds copper in the relatively unstable ML complex, plays this initiating role. All the techniques used do effectively concur to demonstrate that, in spite of the 8-membered chelate ring required for multidentate coordination, the Lys 3 amino group induces the chelation process that gives rise to the MLH −1 species, probably through the (Ala 2—Lys 3) site. Then, the classical scheme prevails with the deprotonation of the amide group at the Lys 3—Ser 4 junction to form MLH −2. Finally, because of the important steric crowding around the metal ion which prevents it from binding to a third deprotonated peptide bond, MLH −3 forms via the deprotonation of the hydroxyl group of the Ser 4 residue. Implications of this particular coordination scheme for the discriminating role of the Lys 3 residue versus thymulin activity are briefly discussed.