Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor
Abstract
:1. Introduction
CaM | Position in CaM | ||||||||
---|---|---|---|---|---|---|---|---|---|
36 | 51 | 71 | 72 | 76 | 109 | 124 | 144 | 145 | |
wt | M | M | M | M | M | M | M | M | M |
L9 | L | L | L | L | L | L | L | L | L |
M36/L8 | M | L | L | L | L | L | L | L | L |
M51/L8 | L | M | L | L | L | L | L | L | L |
M71/L8 | L | L | M | L | L | L | L | L | L |
M72/L8 | L | L | L | M | L | L | L | L | L |
M76/L8 | L | L | L | L | M | L | L | L | L |
M144/L8 | L | L | L | L | L | L | L | M | L |
M145/L8 | L | L | L | L | L | L | L | L | M |
M36,M51/L7 | M | M | L | L | L | L | L | L | L |
M71,M72,M76/L6 | L | L | M | M | M | L | L | L | L |
M109,M124/L7 | L | L | L | L | L | M | M | L | L |
M144,M145/L7 | L | L | L | L | L | L | L | M | M |
L2 | M | M | M | M | M | M | M | L | L |
2. Results
2.1. Ca2+-Dependence of CaM Stimulation of EF
2.2. Regulation of EF by the Degree of Met Oxidation in CaM
2.3. Regulation of EF Activation by Native CaM-mut with Met to Leu Substitutions
2.4. Regulation of EF Activation by Site-Specific N- and C-Terminal Met Oxidation
CaM | AC Activity with 10 μM CaM-wt/CaM-mut [%] | |
---|---|---|
Oxidized | Oxidized | |
wt | 3.3 ± 2.1 *** | 86.3 ± 6.9 *** |
L9 | 100.3 ± 15.9 | 100.6 ± 5.2 |
M36/L8 | 95.7 ± 7.5 | 97.7 ± 5.0 |
M51/L8 | 85.9 ± 15.7 | 99.1 ± 10.0 |
M71/L8 | 91.2 ± 3.0 | 99.1 ± 4.7 |
M72/L8 | 94.9 ± 7.8 | 101.3 ± 5.2 |
M76/L8 | 84.7 ± 12.8 | 102.7 ± 10.4 |
M144/L8 | 101.5 ± 15.4 | 95.7 ± 2.4 |
M145/L8 | 104.9 ± 16.6 | 100.8 ± 16.7 |
M36,M51/L7 | 90.9 ± 9.7 | 108.4 ± 7.2 |
M71,M72,M76/L6 | 86.4 ± 12.5 | 111.0 ± 7.1 |
M109,M124/L7 | 82.4 ± 11.4 | 106.9 ± 6.9 |
M144,M145/L7 | 96.2 ± 6.7 | 96.3 ± 3.9 |
L2 | 74.8 ± 5.8 ** | 91.3 ± 7.0 |
CaM | pEC50 | ||
---|---|---|---|
Native | Oxidized | MsrA-treated | |
wt | 7.61 ± 0.05 | - | 6.35 ± 0.09 *** |
L9 | 7.40 ± 0.10 | 7.31 ± 0.13 ** | 6.96 ± 0.05 *** |
M36/L8 | 6.81 ± 0.06 | 6.50 ± 0.06 *** | 6.29 ± 0.04 *** |
M51/L8 | 6.96 ± 0.09 | 6.83 ± 0.14 ** | 6.50 ± 0.07 *** |
M71/L8 | 6.83 ± 0.08 | 6.35 ± 0.05 *** | 6.35 ± 0.05 *** |
M72/L8 | 7.05 ± 0.07 | 6.75 ± 0.07 *** | 6.57 ± 0.05 *** |
M76/L8 | 6.81 ± 0.10 | 6.47 ± 0.10 *** | 6.37 ± 0.06 *** |
M144/L8 | 7.54 ± 0.11 | 7.08 ± 0.10 *** | 6.86 ± 0.04 *** |
M145/L8 | 7.73 ± 0.05 | 7.14 ± 0.10 *** | 6.97 ± 0.07 *** |
M36,M51/L7 | 6.88 ± 0.07 | 6.19 ± 0.06 *** | 6.29 ± 0.04 *** |
M71,M72,M76/L6 | 6.94 ± 0.06 | 6.30 ± 0.07 *** | 6.27 ± 0.04 *** |
M109,M124/L7 | 6.95 ± 0.05 | 5.90 ± 0.06 *** | 6.35 ± 0.04 *** |
M144,M145/L7 | 7.56 ± 0.09 | 6.52 ± 0.06 *** | 6.92 ± 0.05 *** |
L2 | 7.40 ± 0.10 | 5.33 ± 0.07 *** | 6.62 ± 0.05 *** |
2.5. Restoration of CaM Stimulation of EF by Msr Catalyzed Reduction of MetSO
3. Discussion
3.1. Impact of Intact C-Terminal Structure of CaM on EF Activation
3.2. Comparison of CaM-EF Interaction to Other CaM-Target Interactions
M a | Membranous AC1 b | Bacillus anthracis AC toxin EF | Bordetella pertussis AC Toxin CyaA c |
---|---|---|---|
36 | High relevance: Single Met in the N-terminal region of CaM enhances AC1 activation and MetSO impairs AC1 activation | Moderate relevance: Important for CaM-EF binding, oxidation decreases CaM-EF affinity; structure: No contact to CaM within the CaM-EF complex | No relevance: Met is accessible to oxidation within the CaM-CyaA complex; oxidation does not prevent binding to CyaA |
51 | High relevance: MetSO impairs AC1 activation | Moderate relevance: Like M36 | No relevance: Like M36 |
71 | Only relevant in combination of oxidized M72, M72 and M76: AC1 activity decreases significantly | Moderate relevance: Like M36 | No relevance: Like M36 |
72 | Like M71 | Moderate relevance: Like M36 | No relevance: Like M36 |
76 | Like M71 | Moderate relevance: Like M36 | No relevance: Like M36 |
109 | High relevance in combination of oxidized M109 and M124: AC1 activity decreases significantly | Low relevance: Oxidation of M109 and M124 does not affect EF activation; structure: Met is in contact with CaM within the CaM-EF complex | High relevance: Crystallographic studies reveal important hydrophobic interactions with CyaA; preservation of this Met from oxidation within the CaM-CyaA complex and prevention of binding to CyaA in oxidized state suggest an involvement in the formation of CaM-CyaA complex |
124 | High relevance: Like M109 | Low relevance: Like M109 | High relevance: Like M109 |
144 | High relevance: Met enhances AC1 activation and MetSO impairs AC1 activation; no altered AC1 activation in combination of two Met or MetSO at position 144 and 145 | High relevance: An intact C-terminal region next to M144 and M145 is sufficient for effective EF activation also upon oxidation of all other Met, important for the high affinity of EF to CaM; molecular modeling: Met is in contact with CaM within the CaM-EF complex | Moderate relevance: Crystallographic studies reveal important hydrophobic interactions with CyaA; Met is accessible to oxidation within the CaM-CyaA complex; site-specific oxidation does not prevent binding to CyaA but in combination with other C-terminal Met oxidation prevents binding to CyaA |
145 | High relevance: Like M144 | High relevance: Like M144 | High relevance: Like M109, additionally: Important for CyaA activation |
4. Experimental Section
4.1. Materials
4.2. Expression and Purification of EF
4.3. Cloning, Expression, and Purification of CaM-wt and CaM-mut
4.4. Oxidation of Met and Reduction of MetSO
4.5. AC Activity Assay
4.6. Statistics
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Lübker, C.; Dove, S.; Tang, W.-J.; Urbauer, R.J.B.; Moskovitz, J.; Urbauer, J.L.; Seifert, R. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor. Toxins 2015, 7, 2598-2614. https://doi.org/10.3390/toxins7072598
Lübker C, Dove S, Tang W-J, Urbauer RJB, Moskovitz J, Urbauer JL, Seifert R. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor. Toxins. 2015; 7(7):2598-2614. https://doi.org/10.3390/toxins7072598
Chicago/Turabian StyleLübker, Carolin, Stefan Dove, Wei-Jen Tang, Ramona J. Bieber Urbauer, Jackob Moskovitz, Jeffrey L. Urbauer, and Roland Seifert. 2015. "Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor" Toxins 7, no. 7: 2598-2614. https://doi.org/10.3390/toxins7072598