Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: Comparison with a mutant protein

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants-P7S and P9A of human Zn₇-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and ¹¹³Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd₇-mutMT-3 were investigated and compared with those of wild-type Cd₇-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasak, M. (1999) Biochemistry 38, 10158] and the well characterized Cd₇-MT-2a from rabbit liver. Similarly to ¹¹³Cd₇-MT-3 the ¹¹³Cd NMR spectrum of ¹¹³Cd₇-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd₄S₁₁ cluster in the α-domain and a Cd₃S₉ cluster in the β-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [¹¹³Cd-¹¹³Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in ¹¹³Cd₇-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd₃S₉ cluster resonances, no such temperature effect was observed in ¹¹³Cd₇-MT-3. To account for the observed NMR features, a dynamic structural model for the β-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in ¹¹³Cd₇-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. The results suggest that specific structural features dictated by the repetitive (Cys-Pro)₂ sequence in the β-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.