STRUCTURAL STABILIZATION OF BOTULINUM NEUROTOXINS BY TYROSINE PHOSPHORYLATION. J.A. ENCINAR(1), A.V. FERRER-MONTIEL(2), A. FERNµNDEZ(1), B.R. DASGUPTA(3), J.A. FERRAGUT(1), M. MONTAL(2) & J.M. GONZµLEZ-ROS(1) (1) DEPT. NEUROCHEMISTRY AND INSTITUTE OF NEUROSCIENCES. UNIVERSITY MIGUEL HERNµNDEZ. 03080. ALICANTE. ESPA¥A. (2) DEPT. BIOLOGY, UNIVERSITY OF CALIFORNIA SAN DIEGO, LA JOLLA, CALIFORNIA 92093-0366. U.S.A. (3) UNIVERSITY OF WISCONSIN, MADISON, WISCONSIN 53076. U.S.A. INTRODUCTION Botulinum neurotoxins (BoNTs), the causative agents of botulism, are a family of bacterial proteins that produce flaccid paralysis in skeletal muscles by blocking Ca2+-evoked exocytosis. BoNTs are proteins of 150 kD, composed of a heavy chain of 100 kD and a light chain of 50 kDa. The light chain is a metalloprotease that cleaves proteins involved in neurosecretion. The in vitro lability of pure BoNTs contrast with their exceptional stability inside the cells, suggesting their potential intracellular modification. Indeed, we recently discovered that tyrosine phosphorylation of BoNTs drastically increases both their catalytic activity and thermal stability (Ferrer-Montiel et al. (1996) J. Biol. Chem. 271:18322-18325). In this communication, we have used FTIR spectroscopy and report that tyrosine phosphorylation of two different serotypes of BoNTs (BoNT A and BoNT E) increases the content of alpha-helix secondary structure as evidenced by analysis of the conformationally-sensitive Amide I band. This secondary structural change is accompanied by an increase in the Amide II band remaining upon exchange with D2O, which further indicates a change in the overall protein structure that produces a markedly different accesibility to the solvent. RESULTS The strong Amide I band, comprising the 1700-1600 cm-1 infrared spectral region, results primarily from stretching vibrations of C=O groups in peptide bonds, the exact frequencies of which are determined by the particular secondary structure adopted by the protein. The information provided by the Amide I band in the original spectrum is limited by the intrinsic widths of the spectral components contributed by the different protein secondary structures, which are usually larger than their frequency separation and thus, result in spectral overlapping. Application of band-narrowing procedures, such as Fourier self-deconvolution or Fourier derivation, allow to visualize the individual components. Figure 1 shows the original and the deconvolved spectra of control and phosphorylated BoNTs samples. The Amide I band region of both BoNT A and BoNT E samples exhibits maxima at approximately 1690, 1980, 1668, 1652, 1640, 1630 and 1615 cm-1. Whereas the 1615 cm-1 component correspond to amino acid side-chain vibration, all the other maxima have been assigned to vibration of the carbonyl group in peptide bonds within different BoNTs secondary structural motifs: the 1630 cm-1 component is assigned to beta-structure, the 1640 cm-1 to random structure, the 1652 cm-1 component to alpha-helix, the 1690 and 1680 cm-1 components to turns, and the 1668 cm-1 component includes contributions from turns as well as from the (0, pi) beta-sheet vibration band. The main changes observed in the Amide I band of the phosphorylated samples seem to refer to an increase in the 1652 cm-1 component assigned to alpha-helix (Figure 1). In order to quantitate the secondary structure in these samples, we have used a maximum entropy method in order to reduce spectral noise and consequently, minimize the errors in the estructural determination. Figure 2A illustrate the results that can be obtained from the band-fitting analysis of the original Amide I band of control and phosphorylated BoNTs samples. Figure 2B summarizes such results and shows that phosphorylation of BoNTs indeed produces a pronounced increase (?20 %) in alpha-helical structure, concomitant with the decrease in both 1668 and 1640 cm-1 bands, which are assigned to turns and random structures, respectively. IR spectra were also taken at progressively higher temperatures to monitor thermal denaturation in the BoNTs samples. There were only minor effects of temperature on the Amide I band in the control samples. However, the temperature- dependent changes were much more noticeable in the phosphorylated samples, causing the appearance of two components at 1618 and 1685 cm-1, which in most other proteins, have been related to aggregation of thermally unfolded regions (see Figure 3). A most dramatic change difference between the infrared spectra of phosphorylated and control spectra is that even after 2 h of exposure to the D2O media, the phosphorylated samples exhibit a prominent residual Amide II band (primarily peptide N-H bending) centered near 1550 cm-1. According to the above observations in the Amide I band, the large proportion of peptide hydrogens in the phosphorylated BoNTs that can not be exchangedly deuterium are likely to corresponds to alpha-helical peptide hydrogens. Moreover, the residual Amide II band in the phosphorylated samples still remains after heating at 70 ºC, indicating that phosphorylation of BoNTs produces a much more stable and compact structure which, remains partly unaccessible to exchange with the solvent, even at the high temperature used in the experiments (Figure 4). CONCLUSIONS 1.- Tyrosine phosphorylation of two different serotypes of BoNTs (BoNT A and BoNT E) increases the contents of alpha-helical secondary structure. 2.- Phosphorylated BoNTs samples exhibit a residual Amide II band remaining upon exchange with D2O, suggesting that a much tighter packing is produced upon phosphorylation. 3.- The above observations partly explain in structural terms the previous report (Ferrer-Montiel et al. (1996) J. Biol. Chem. 271:18322-18325) on an increase in catalytic activity and thermal stability caused by phosphorylation. REFERENCES - Arrondo, J.L.R., Mantsch, H.H., Mullner, N., Pikula, S. and Martonosi, A. (1987) J. Biol. Chem. 262: 9037-9043. - Echabe, I., Encinar, J.A. and Arrondo, J.L.R. (1997) Biospectroscopy (In press). - Ferrer-Montiel, A.-V., Canaves, J.M., DasGusta, B.R., Wilson, M.C. and Montal, M. (1996) J. Biol. Chem. 271: 18322-18325. - Surewicz, W.K., Leddy, J.J. and Mantsch, H.H. (1990) Biochemistry 29: 8106- 8111.