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The A sub-unit can be broken down into A1 and A2 units. This break down occurs when a trypsin sensitive region, residues 248-251, is cleaved. This cleavage results in the only remaining link being a <scene name='68/687332/Disulfide_bridge/1'>disulfide bridge (1R4Q)</scene> between cysteine 242 and cysteine 261. If the disulfide bridge is reduced, the A1 and A2 fragments can completely separate. Interestingly, the disulfide bridge blocks the active site of the A sub-unit, so the A sub-unit is not enzymatically active unless it has been cleaved<ref name ="Fraser">Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200</ref>.
The A sub-unit can be broken down into A1 and A2 units. This break down occurs when a trypsin sensitive region, residues 248-251, is cleaved. This cleavage results in the only remaining link being a <scene name='68/687332/Disulfide_bridge/1'>disulfide bridge (1R4Q)</scene> between cysteine 242 and cysteine 261. If the disulfide bridge is reduced, the A1 and A2 fragments can completely separate. Interestingly, the disulfide bridge blocks the active site of the A sub-unit, so the A sub-unit is not enzymatically active unless it has been cleaved<ref name ="Fraser">Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200</ref>.
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<scene name='68/687332/B_subunit/1'>The B sub-unit (2xsc)</scene> is a symmetrical pentamer that resembles a star and can be divided into five identical monomers. Each monomer is 69 amino acids in length. The purpose of the B sub-unit is to bind to globotriaosylceramide (GB3) which is a glycosphingolipid that is found on the lipid rafts of endothelial cells. Each monomer of the B sub-unit has three binding sites for GB3, binding to the <scene name='68/687332/Gb3_bound_to_subunit_b/1'>carbohydrate portion of GB3 (1cqf)</scene>. The affinity between the B sub-unit and GB3 is incredibly low, and is actually one of the lowest recorded affinities for carbohydrate-protein interaction<ref>Jacobson, J. M., Yin, J., Kitov, P. I., Mulvey, G., Griener, T. P., James, M. N. G., … Bundle, D. R. (2014). The crystal structure of Shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. Journal of Biological Chemistry, 289(2), 885–894. doi:10.1074/jbc.M113.518886</ref>. Some studies have shown that the level of saturation and length of the fatty acid on GB3 affects the strength of its interaction with the B sub-unit. Since the B sub-unit of stx has fifteen binding sites though, it has an incredibly high avidity<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>.
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<scene name='68/687332/B_subunit/1'>The B sub-unit (2xsc)</scene> is a symmetrical pentamer that resembles a star and can be divided into five identical monomers. Each monomer is 69 amino acids in length. The purpose of the B sub-unit is to bind to globotriaosylceramide (GB3) which is a glycosphingolipid that is found on the lipid rafts of endothelial cells. Each monomer of the B sub-unit has three binding sites for GB3, binding to the <scene name='68/687332/Gb3_bound_to_subunit_b/1'>carbohydrate portion of GB3 (1cqf)</scene>. The B sub-unit has a millimolar affinity for GB3, and is actually one of the lowest recorded affinities for carbohydrate-protein interaction<ref>Jacobson, J. M., Yin, J., Kitov, P. I., Mulvey, G., Griener, T. P., James, M. N. G., … Bundle, D. R. (2014). The crystal structure of Shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. Journal of Biological Chemistry, 289(2), 885–894. doi:10.1074/jbc.M113.518886</ref>. Some studies have shown that the level of saturation and length of the fatty acid on GB3 affects the strength of its interaction with the B sub-unit. Since the B sub-unit of stx has fifteen binding sites though, it significantly increases the strength of the interaction.<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>.
== Function ==
== Function ==
The pathway of stx entering a cell begins with the B sub-unit’s binding to GB3. Once this occurs, the A sub-unit disconnects from the B sub-unit and enters the cell through endocytosis. Using retrograde transport the A sub-unit passes through the Golgi apparatus and the rough endoplasmic reticulum<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref> In the rough endoplasmic reticulum, the A sub-unit is cleaved into two parts, A1 and A2, through the cleavage of trypsin sensitive residues and the reduction of a disulfide bridge. A2 is degraded, but A1 freely enters the cytosol<ref name="Sandvig">Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943</ref>. Once in the cytosol, A1 acts as an N-glycosidase, hydrolyzing bonds that link sugars. With this enzymatic activity, A1 removes an adenine from the alpha-sarcin loop in the 28S RNA of the 60S ribosomal sub-unit<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>. The removal of the adenine prevents elongation factors from associating with the ribosomal sub-unit. Without elongation factors, the ribosome can no longer synthesize proteins, leading to cell death<ref name="Melton">Melton-Cesla, A. (2012). Shiga toxin classification structure and function. Changes, 29(2), 997–1003</ref>.
The pathway of stx entering a cell begins with the B sub-unit’s binding to GB3. Once this occurs, the A sub-unit disconnects from the B sub-unit and enters the cell through endocytosis. Using retrograde transport the A sub-unit passes through the Golgi apparatus and the rough endoplasmic reticulum<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref> In the rough endoplasmic reticulum, the A sub-unit is cleaved into two parts, A1 and A2, through the cleavage of trypsin sensitive residues and the reduction of a disulfide bridge. A2 is degraded, but A1 freely enters the cytosol<ref name="Sandvig">Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943</ref>. Once in the cytosol, A1 acts as an N-glycosidase, hydrolyzing bonds that link sugars. With this enzymatic activity, A1 removes an adenine from the alpha-sarcin loop in the 28S RNA of the 60S ribosomal sub-unit<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>. The removal of the adenine prevents elongation factors from associating with the ribosomal sub-unit. Without elongation factors, the ribosome can no longer synthesize proteins, leading to cell death<ref name="Melton">Melton-Cesla, A. (2012). Shiga toxin classification structure and function. Changes, 29(2), 997–1003</ref>.

Current revision

This Sandbox is Reserved from 15-Jan-2015, through 30-May-2015 for use in the course "Biochemistry" taught by Jason Telford at the Maryville University. This reservation includes Sandbox Reserved 977 through Sandbox Reserved 986.
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Shiga Toxin

Crystal Structure for Shiga Toxin (1R4Q)

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References

  1. ↑ 1.0 1.1 Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200
  2. ↑ 2.0 2.1 Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943
  3. ↑ Jacobson, J. M., Yin, J., Kitov, P. I., Mulvey, G., Griener, T. P., James, M. N. G., … Bundle, D. R. (2014). The crystal structure of Shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. Journal of Biological Chemistry, 289(2), 885–894. doi:10.1074/jbc.M113.518886
  4. ↑ 4.0 4.1 4.2 4.3 Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web
  5. ↑ Zaidi, Mussaret Bano, and Teresa Estrada-García. "Shigella: A Highly Virulent and Elusive pathogen." Current Tropical Medicine Reports 1.2 (2014): 81-87
  6. ↑ Kaper, James B. and Alison D. O’Brien. “Overview and Historical Perspectives.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0028–2014. PMC. Web
  7. ↑ Krüger, Alejandra, and Paula María Alejandra Lucchesi. "Shiga toxins and stx-phages: highly diverse entities." Microbiology (2014): mic-0
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