The characterization of the structure of the active reactive site loop of alpha1-antitrypsin by multidimensional NMR methods

• Main Author: Patera, Alexandra Sacha
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1994
• Subjects: Biochemistry
• ISBN: 9798208813508

Serpins are a family of related proteins most of which act as specific inhibitors of serine proteinases. The details of the inhibitory mechanism of serpins are not well understood, in part, due to little direct structural information about the active form of any serpin. The application of multidimensional NMR methods towards the structural determination of the reactive site loop (RSL) of $\alpha$1-antitrypsin (AT), in its active conformation, is reported. Elastase inhibitory activity of AT has been conferred to the cytokine interleukin-1$\beta$ (IL-1$\beta$) by replacing residues E50-E51-S52-N53 with the ten amino acid serpin RSL segment, EAIPMSIPPE. The IL-1$\beta$ $\beta$-barrel portion of the chimeric protein, AT/IL$\sb{50-53}$, is structurally indistinguishable from the wild type IL-1$\beta$. Sharp new $\sp1$H resonances appear in AT/IL$\sb{50-53}$ spectra which correspond to the ten amino acid insertion. The narrow linewidths of these resonances suggest that the loop is mobile relative to the rest of the protein. However, the three prolines do not appear to undergo cis/trans isomerism. Residues E-A-I extend the $\beta$-strand of the native IL-1$\beta$ V41-G49, while M-S-I adopt a more turn-like conformation. Computed $\phi$ and $\chi\sb1$ angles from J-coupling constants and interresidue NOEs were used to constrain the backbone of the elastase inhibitory loop for dynamical simulated annealing calculations. The first generation of computed structures of the AT reactive site loop does not appear to adopt a defined secondary structure such as the helix in ovalbumin (Stein et al., 1990) or the "distorted" helix of the recent structure of the ACh/At mutant (Wei et al., 1994). The preliminary characterization of the solution structure of the active $\alpha$1-antitrypsin reactive site loop is described.