Evidence for a dynamic structure of human neuronal growth inhibitory factor and for major rearrangements of its metal -: Thiolate clusters

Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn-7- and Cd-7-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and Cd-113 NMR. The CD and Cd-113 NMR studies of recombinant Me-7-GIF confirmed the existence of distinct Me4S11- and Me3S9-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The Cd-113 NMR studies of recombinant Cd-113(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven Cd-113 signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [Cd-113-Cd-113] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [Cd-113, Cd-113] COSY. On the basis of a close correspondence in chemical shift positions of the Cd-113 signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of Cd-113(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasak, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd4S11-cluster in the a-domain of Cd-113(7)-GIF. Consequently, the remaining three Cd-113 signals at 664, 651, and 644 ppm originate from a Me3S9 cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (similar to 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of Cd-113(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.