Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Expression and Purification
2.2. Small-Angle X-ray Scattering (SAXS)
2.3. Ab Initio Molecular Shape Analysis
2.4. EPAC1 Homology Models for Rigid-Body Analysis
2.5. Rigid-Body Analysis
2.6. Polydispersity and Conformational Ensemble Analyses of EPAC1
3. Results
3.1. In Solution the apo-EPAC1 NTD Blocks the Effector Binding Site
3.2. The Solution Structure of apo-EPAC1 is a Dynamic Mixture of Closed and Extended States
3.3. In Solution, cAMP-Bound EPAC1 Forms Dimers
3.4. The Solution Structure of the EPAC1:cAMP:Rap1 Ternary Complex
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Appendix A
SAXNS_ES Buffer Subtraction, MW Calibration, and MW Calculation
References
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apo-EPAC1 | cAMP: EPAC1 | cAMP: EPAC1: Rap1b | |
---|---|---|---|
q-range (Å−1) | 0.010–0.30 | 0.012–0.30 | 0.012–0.30 |
Concentration (mg/mL) | 0.5 | 2.7 | 1.0 |
Time (h) | |||
Sample | 6 | 2 | 2.5 |
Buffer | 6 | 3 | 11.5 |
Rg (Å) | 33.7 ± 0.5 | 53.2 ± 0.7 | 40.5 ± 0.6 |
Dmax (Å) | 110 | 185 | 142 |
MW (kDa) | 100 | 100 | 119 |
MW(Io) (kDa) {N} | 104 {1.0} | 178 {1.8} | 98 {0.8} |
MW(Porod) (kDa) {N} | 113 {1.1} | 240 {2.4} | 124 {1.0} |
MW(Rambo) (kDa) {N} | 97.8 {1.0} | 218 {2.2} | 126 {1.1} |
DAMMIF NSD (10) | 0.8 ± 3 | 1.0 ± 8 | 0.9 ± 1 |
SASRES(Å) | 40 ± 3 | 14 ± 2 | 42 ± 3 |
Rigid Body Fitting | |||
HM Crysol fit (X2) | 1.3 | NA | NA |
CORAL fit (X2) | 1.0 | 1.0 | 1.0 |
CORAL Model Rg (Å) | 32.7 | 41.5 1 | 40.5 |
EOM:(X2) | 1.0 | 1.1 | 0.7 |
Rg [FWHM] 2 {Area} | |||
Peak (closed) | 32.9 [0.8] 2 {86%} | 0 | 0 |
Peak (extended) | 38.5 [0.9] 2 {13%} | 0 | 0 |
Peak (ternary) | 0 | 0 | 40.7 [0.7] 2 {100%} |
Peak (dimer) | 0 | 51 [3] 2 {100%} | 0 |
SASDB-id | SASDCQ6 | SASDCR6 | SASDCS6 |
Analysis | Software: | Analysis | Software: |
Image Data-processing: | SAXSLAB (Rigaku) | Buffer Subtraction: | SAXNS-ES 3 [45] |
Data-analysis: | PRIMUS [48] | Real Space P(r): | GNOM [48], BayesApp [49] |
Ab initio Models: | DAMMIF [50] | Molecular Weight: | SAXSMOW [51] |
Rigid Body Refinement: | CORAL [52], BUNCH | Ensemble Analysis: | EOM [53], MES [54] |
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White, M.A.; Tsalkova, T.; Mei, F.C.; Cheng, X. Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology. Cells 2020, 9, 35. https://doi.org/10.3390/cells9010035
White MA, Tsalkova T, Mei FC, Cheng X. Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology. Cells. 2020; 9(1):35. https://doi.org/10.3390/cells9010035
Chicago/Turabian StyleWhite, Mark Andrew, Tamara Tsalkova, Fang C. Mei, and **aodong Cheng. 2020. "Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology" Cells 9, no. 1: 35. https://doi.org/10.3390/cells9010035