MODULATION OF DIMERIZATION BY RESIDUES DISTANT FROM THE INTERFACE IN BOVINE NEUROPHYSIN-II

The crystal structure of bovine neurophysin-II in its liganded state ( Chen et al. [1991] Proc. Natl. Acad. Sci. USA 88, 4240-4244) indicates that the 16 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the mono mer-monomer interface. Cleavage of the 16 sequence by Staphylococcus p rotease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 16 sequence in the unliganded state were obtained by NMR and related bio physical comparisons of the native and des-1-6 proteins. NMR spectra d emonstrated that the environment and/or conformation of residues in th e 1-6 sequence differed in liganded and unliganded states. Additionall y, the unliganded des-1-6 protein exhibited a dimerization constant fo ur to five times that of the native protein, potentially accounting fo r the observation that its peptide affinity was also increased. NMR st udies further indicated that the increased dimerization constant of th e des-1-6 protein correlated with the presence in the native protein o f two isoenergetic forms of the monomer, in contrast to only a single form in the des-1-6 protein, as evidenced by signals from an internal dimerization-sensitive alpha-proton. Thus, the 16 sequence reduces the dimerization constant by stabilization of an alternative monomer conf ormation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des-1-6 protein with an inter nal clip after binding site residue Glu-47, the clip presumably breaki ng the short 3,10 helix that most directly connects the interface to t he binding site. This product, although unable to bind peptide, retain ed the dimerization constant of the des-l-6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the e ffects of the 1-6 sequence. A model is proposed in which the 16 sequen ce stabilizes the second conformation of the unliganded monomer via in teractions affecting the loop region that separates the two neurophysi n domains and which has been shown to influence neurophysin self-assoc iation. (C) Munksgaard 1997.