Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation

Science

Volumn 284, Issue 5416, 1999, Pages 955-957

  Ren, Jianhua ^a   Kachel, Kelli ^a   Kim, Hyun ^a   Malenbaum, Susan E ^a,c   Collier, R John ^b   London, Erwin ^a  

^a STONY BROOK UNIVERSITY   (United States)

^b HARVARD MEDICAL SCHOOL   (United States)

^c AARON DIAMOND AIDS RESEARCH CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHAPERONE; DIPHTHERIA TOXIN;

ALPHA CHAIN; ARTICLE; BETA CHAIN; CONFORMATION; CORYNEBACTERIUM DIPHTHERIAE; INTRACELLULAR TRANSPORT; NONHUMAN; PH; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN FOLDING; PROTEIN PROTEIN INTERACTION; PROTEIN TRANSPORT;

ANTIBODIES; APOPROTEINS; BORON COMPOUNDS; CATALYTIC DOMAIN; DIPHTHERIA TOXIN; ENERGY TRANSFER; FLUORESCENCE; HYDROGEN-ION CONCENTRATION; LACTALBUMIN; MEMBRANES, ARTIFICIAL; MYOGLOBIN; PEPTIDE FRAGMENTS; PROTEIN CONFORMATION; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; PROTEINS; SERUM ALBUMIN;

CORYNEBACTERIUM DIPHTHERIAE; POSIBACTERIA;

EID: 0033532355     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5416.955     Document Type: Article

References (32)

1. S. Choe et al., Nature 357, 216 (1992).
2. E. London, Biochim. Biophys. Acta 1113, 25 (1992); E. London, Mol. Microbiol. 6, 3277 (1992).
3. E. London, Biochim. Biophys. Acta 1113, 25 (1992); E. London, Mol. Microbiol. 6, 3277 (1992).
4. J.-M. Zhao and E. London, J. Biol. Chem. 263, 15369 (1988).
5. P. O. Falnes et al., ibid. 269, 8402 (1994).
6. P. O. Falnes and S. Olsnes, ibid. 270, 20787 (1995).
7. E. Papini et al., Eur. J. Biochem. 169, 637 (1987).
8. Y. Wang et al., J. Biol. Chem. 272, 25091 (1997).
9. K. Kachel, J. Ren, R. J. Collier, E. London, ibid. 273, 22950 (1998).
10. K. J. Oh et al., Science 273, 810 (1996).
11. P. J. Sadler and A. Tucker, Eur. J. Biochem. 212, 811 (1993); E. Sulkowski, M. Madajewicz, D. Doyle, in Affinity Chromatography and Biological Recognition, I. H. Irwin, M. Wilchek, P. Indu, Eds. (Academic Press, New York, 1983), pp. 489-494; W. Pfiel, V. E. Bychkova, O. B. Ptitsyn, FEBS Lett. 198, 287 (1986); Y. Goto and A. L. Fink J. Mol. Biol. 214, 803 (1990).
12. P. J. Sadler and A. Tucker, Eur. J. Biochem. 212, 811 (1993); E. Sulkowski, M. Madajewicz, D. Doyle, in Affinity Chromatography and Biological Recognition, I. H. Irwin, M. Wilchek, P. Indu, Eds. (Academic Press, New York, 1983), pp. 489-494; W. Pfiel, V. E. Bychkova, O. B. Ptitsyn, FEBS Lett. 198, 287 (1986); Y. Goto and A. L. Fink J. Mol. Biol. 214, 803 (1990).
13. P. J. Sadler and A. Tucker, Eur. J. Biochem. 212, 811 (1993); E. Sulkowski, M. Madajewicz, D. Doyle, in Affinity Chromatography and Biological Recognition, I. H. Irwin, M. Wilchek, P. Indu, Eds. (Academic Press, New York, 1983), pp. 489-494; W. Pfiel, V. E. Bychkova, O. B. Ptitsyn, FEBS Lett. 198, 287 (1986); Y. Goto and A. L. Fink J. Mol. Biol. 214, 803 (1990).
14. P. J. Sadler and A. Tucker, Eur. J. Biochem. 212, 811 (1993); E. Sulkowski, M. Madajewicz, D. Doyle, in Affinity Chromatography and Biological Recognition, I. H. Irwin, M. Wilchek, P. Indu, Eds. (Academic Press, New York, 1983), pp. 489-494; W. Pfiel, V. E. Bychkova, O. B. Ptitsyn, FEBS Lett. 198, 287 (1986); Y. Goto and A. L. Fink J. Mol. Biol. 214, 803 (1990).
15. S. Banuelos and A. Muga, J. Biol. Chem. 270, 29910 (1995); J. W. Lee and H. Kim, Arch. Biochem, Biophys. 297, 354 (1992).
16. S. Banuelos and A. Muga, J. Biol. Chem. 270, 29910 (1995); J. W. Lee and H. Kim, Arch. Biochem, Biophys. 297, 354 (1992).
17. We confirmed that there was partial unfolding of α-lactalbumin, apomyoglobin, and HSA by both circular dichroism and fluorescence. Partial unfolding can be detected for apomyoglobin by the decrease in the (absolute values of) ellipticity at 222 nm [F. M. Hughson, D. Barrick, R. L. Baldwin Biochemistry 30, 4113 (1991)], for α-lactalbumin by the decrease in ellipticity at 280 nm [J. J. Ewbank and T. E. Creighton, Nature 350, 518 (1991)], and for HSA by an unusual blue shift in Trp emission [J. Y. Lee and M. Hirose, J. Biol. Chem. 267, 14753 (1992)]. Relative to the signal in solution at neutral pH, we found that in the presence of DOPG/ DOPC vesicles at pH 4.1 there was a 20% decrease in the absolute value of the ellipticity of apomyoglobin at 222 nm, a 33% reduction of α-lactalbumin ellipticity at 280 nm, and a 13-nm blue shift in HSA Trp emission wavelength. We also found that in the presence of DOPC vesicles, the blue shift in HSA fluorescence was 5 nm greater upon reduction of HSA by DTT.
18. We confirmed that there was partial unfolding of α-lactalbumin, apomyoglobin, and HSA by both circular dichroism and fluorescence. Partial unfolding can be detected for apomyoglobin by the decrease in the (absolute values of) ellipticity at 222 nm [F. M. Hughson, D. Barrick, R. L. Baldwin Biochemistry 30, 4113 (1991)], for α-lactalbumin by the decrease in ellipticity at 280 nm [J. J. Ewbank and T. E. Creighton, Nature 350, 518 (1991)], and for HSA by an unusual blue shift in Trp emission [J. Y. Lee and M. Hirose, J. Biol. Chem. 267, 14753 (1992)]. Relative to the signal in solution at neutral pH, we found that in the presence of DOPG/ DOPC vesicles at pH 4.1 there was a 20% decrease in the absolute value of the ellipticity of apomyoglobin at 222 nm, a 33% reduction of α-lactalbumin ellipticity at 280 nm, and a 13-nm blue shift in HSA Trp emission wavelength. We also found that in the presence of DOPC vesicles, the blue shift in HSA fluorescence was 5 nm greater upon reduction of HSA by DTT.
19. We confirmed that there was partial unfolding of α-lactalbumin, apomyoglobin, and HSA by both circular dichroism and fluorescence. Partial unfolding can be detected for apomyoglobin by the decrease in the (absolute values of) ellipticity at 222 nm [F. M. Hughson, D. Barrick, R. L. Baldwin Biochemistry 30, 4113 (1991)], for α-lactalbumin by the decrease in ellipticity at 280 nm [J. J. Ewbank and T. E. Creighton, Nature 350, 518 (1991)], and for HSA by an unusual blue shift in Trp emission [J. Y. Lee and M. Hirose, J. Biol. Chem. 267, 14753 (1992)]. Relative to the signal in solution at neutral pH, we found that in the presence of DOPG/ DOPC vesicles at pH 4.1 there was a 20% decrease in the absolute value of the ellipticity of apomyoglobin at 222 nm, a 33% reduction of α-lactalbumin ellipticity at 280 nm, and a 13-nm blue shift in HSA Trp emission wavelength. We also found that in the presence of DOPC vesicles, the blue shift in HSA fluorescence was 5 nm greater upon reduction of HSA by DTT.
20. Ovalbumin and lysozyme remain folded at moderately low pH and tend to undergo unfolding transitions without MG intermediates [F. Ahmad and A. Salahuddin, Biochim. Biophys. Acta 576, 333 (1979); P. Haezebrouck et al., J. Mol. Biol. 246, 382 (1995)]. Retention of tight dansyl binding demonstrated that anti-dansyl IgG remained folded at low pH (M. Rosconi and E. London, data not shown).
21. Ovalbumin and lysozyme remain folded at moderately low pH and tend to undergo unfolding transitions without MG intermediates [F. Ahmad and A. Salahuddin, Biochim. Biophys. Acta 576, 333 (1979); P. Haezebrouck et al., J. Mol. Biol. 246, 382 (1995)]. Retention of tight dansyl binding demonstrated that anti-dansyl IgG remained folded at low pH (M. Rosconi and E. London, data not shown).
22. There is residual antibody binding because it appears that, at most, half of the T domain molecules convert to the TM state (7, 8).
23. We found that the T domain acquires the TM conformation in large unilamellar vesicles (LUV) (J. Sharpe and E. London, unpublished observations). Thus, the possibility that fusion of small unilamellar vesicles (SUV) into LUV by MC proteins affected T domain conformation was of concern. However, although Sepharose 4B-CL size fractionation of samples containing SUV to which T domain and apomyoglobin were added indicated some vesicle fusion (or aggregation), T domain molecules remaining in SUV-containing fractions still appeared to exhibit the blue shift in bimane fluorescence characteristic of the TM state (data not shown).
24. -1.
25. This was of concern because MG proteins bind to lipids at low pH (11). The absence of nonspecific quenching was confirmed by experiments that showed no significant energy transfer from a membrane-inserted, bimane-labeled polyleucyl peptide [J. Ren, S. Lew, Z. Wang, E. London, Biochemistry 36, 10213 (1997)] to rhodamine-labeled HSA (data not shown). It is also noteworthy that addition of unlabeled MG proteins did not result in dissociation of the rhodamine-labeled HSA from the membrane (data not shown).
26. Comparing the sequence of the A chain to that of other proteins that affect T domain revealed no strong similarities (data not shown).
27. These experiments have not defined the maximum degree of unfolding (or the minimum) that would still allow recognition by the T domain. In this regard it is interesting that we have found that a 25-residue polyalanyl polypeptide that is only partly helical in solution [L-P. Liu, S.-C. Li, N. K. Goto, C. M. Deber, Biopolymers 39, 465 (1996)] can trigger the P to TM conformational change.
28. J. C. Reed, Nature 387, 773 (1997).
29. A. Weidlocha et al., EMBO J. 11, 4835 (1992).
30. 455 differ in Fig. 2 and Table 2 because fluorimeters with different wavelength sensitivities were used.
31. Anti-BODIPY IgG is folded and active at pH 4.1 [J. Ren, R. J. Collier, J. C. Sharpe, E. London, Biochemistry 38, 976 (1999)].
32. Supported by National Institutes of Health grants GM 39186 (E.L.) and AI 22021 (R.J.C.).