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 met allothionein-3 (MT-3), impairs the survival and the neurite formation of cu ltured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and si ngle mutants P7S and P9A of human Zn-7-MT-3 were generated, and their effec ts on the biological activity and the structure of the protein were examine d. 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), magneti c CD (MCD), and Cd-113 NMR spectroscopy, the structural features of the met al-thiolate clusters in the double mutant Cd-7-mutMT-3 were investigated an d compared with those of wild-type Cd-7-MT-3 [Faller, P., Hasler, D. W., Ze rbe, O., Klauser, S., Winge, D. R., and Vasak, M. (1999) Biochemistry 38, 1 0158] and the well characterized Cd-7-MT-2a from rabbit liver. Similarly to Cd-113(7)-MT-3 the Cd-113 NMR spectrum of Cd-113(7)-mutMT-3 at 298 K revea led four major and three minor resonances (approximately 20% of the major o nes) between 590 and 680 ppm, originating from a Cd4S11 cluster in the alph a -domain and a Cd3S9 cluster in the beta -domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all re sonances showed the absence of resolved homonuclear [Cd-113-Cd-113] couplin gs and large apparent line widths (between 140 and 350 Hz). However, wherea s in Cd-113(7)-mutMT-3 the temperature rise to 323 K resulted in a major re covery of the originally NMR nondetectable population of the Cd3S9 cluster resonances, no such temperature effect was observed in Cd-113(7)-MT-3. To a ccount for the observed NMR features, a dynamic structural model for the be ta -domain is proposed, which involves a folded and a partially unfolded st ate. 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 Cd-113(7)-MT-3 tak es place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constant s of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta -domain of MT-3 and not i ts altered metal binding affinity compared to MT-1/MT-2 isoforms are respon sible for the biological activity of this protein.