Orthopaedic Research Laboratory Alumni Council

Alumni Laboratories

Visit the research laboratories of the alumni of Dr. Woo.

Board of Directors

Richard Debski, Ph.D. President
Caroline Wang, M.S. Secretary
Jamie Pfaeffle, M.D., Ph.D. Treasurer
Doug Boardman, M.D.
Thay Lee, Ph.D.
Patrick McMahon, M.D.
Karen Ohland, M.S.
Christos Papgeorgiou, M.D.
Masataka Sakane, M.D.
Sven Scheffler, M.D.
Jennifer Wayne, Ph.D.

2007 Mr. & Mrs. Kwok-Chong Woo Research Grant

Jonathan Suever
University of Alabama at Birmingham

Molecular Insight into the Apoptotic Pathway of Osteoclasts Using Molecular Dynamic Simulations


Jonathan with his advisor, Dr. Yuhua Song

Final Report

Introduction

Osteoporosis is a musculoskeletal disease characterized by a decreased bone density that currently affects 10 million individuals in the United States and is tied to incidents costing an estimated $18 billion annually [1]. One therapeutic approach for the reversal of the symptoms of osteoporosis is stimulation of osteoclast apoptosis. This method attempts to reestablish the careful balance between bone formation and bone resorption required for the maintenance of proper bone density [2]. One apoptotic pathway that has been shown to regulate apoptosis in osteoclasts is the Fas-mediated signaling pathway. Understanding the molecular mechanisms of the signaling pathway is important to identify strategies and targets for the treatment of osteoporosis.

The focus of this investigation was the initial stage of Fas-mediated apoptosis; the binding of Fas to Calmodulin (CaM). Fas is a six alpha-helix protein that interacts with a variety of targets including CaM and the Fas-associated death domain (FADD). CaM is a dumbbell-shaped protein critical for the regulation of a wide range of calcium-dependent cellular processes. It has been shown that the first and second helices of Fas bind with calcium-bound CaM to regulate osteoclast apoptosis.

A point mutation of Fas (V254N) has been shown to decrease the ability of Fas to bind with CaM and impairs the Fas-mediated signaling pathway. Conversely, by removing the last 15 amino acids of the C-terminal of Fas, enhanced binding was exhibited between Fas and CaM [3]. It is hypothesized that the binding of CaM to Fas, and its mutations, alters the structure of Fas, thus affecting its ability to bind with other proteins in the apoptotic signaling pathway.

In this study, molecular dynamics simulations were used to observe the molecular-level interactions between CaM and various Fas mutations.The data was used to analyze the physical and thermodynamic properties of the CaM/Fas complexes. With the information from this study, the function of CaM in the regulation of Fas-mediated apoptosis can be better understood.

Materials & Methods

In order to make the protein complexes to be studied, three dimensional protein crystal structures were obtained from the protein databank for Fas and calcium-bound CaM. Two docking utilities, ZDOCK and RDOCK, were used to create the initial complex to be subjected to the molecular dynamics simulations. The programs selected the most probable complex based upon protein geometry, desolvation, electrostatics, and energy calculations. These complexes were then subjected to molecular dynamics (MD) simulations using the AMBER software package. The protein-protein interactions were simulated for a 30ns time with a time step of 2 fs. In addition to the complexes, each of the proteins and mutations were run through the MD simulations individually as a control group.

The trajectories obtained from the MD simulations were subjected to analyses that determined the binding free energy of the system as well as the changes in the conformation of the protein complex.

Results & Discussion [4]

With the calculation of the binding free energy of the protein complexes, it was seen that the overall binding free energy of the CaM/Fas complex converged after the first 15 ns of the simulation (Figure 1). Using the last 15 ns of the trajectory for analyses, the average binding free energy compared to the results of previous experimental studies [3 ].

Figure 1: Binding free energy of the complexes of CaM and Fas over the 30 ns molecular dynamics simulations with the exclusion of the entropy term.

The simulation results showed that the V254N point mutation exhibited a binding free energy increase of 10.07 kcal/mol (P < 0.05) as compared to the wildtype indicating a decreased binding affinity for the CaM/Fas complex; while the c-terminal deletion had a decreased binding free energy of 20.59 kcal/mol (P < 0.05) compared to the wildtype implying an increase of the binding affinity for the formation of the CaM/Fas complex. The binding free energy, as calculated from the simulation data, was consistent with the binding affinity trends that were seen in biochemical binding studies. After the MD simulation data were verified against the known trends in binding affinity, further analyses was performed to look at the changes in conformation of both Fas and CaM.

To observe the movement of each of the amino acids throughout the course of the simulation, the RMSF (root mean squared fluctuation) was computed for each of the systems. It was seen that the formation of the CaM/Fas complex resulted in a stabilization of the structures of both CaM and Fas as compared to the control simulations for each of the proteins. For the complex formed with the V254N mutation of Fas, there were large fluctuations of Fas on/near its binding site with CaM indicating a structural destabilization of V254N Fas (residues 231-270) (Figure 2). For the c-terminal mutation, there was a lesser degree of conformational fluctuation and an overall stabilization of the structure of Fas. The conformational changes of Fas as a result of the V254N and C-terminal mutations showed that these mutations might contribute to the binding of Fas to CaM seen in previous experiments [3].


Figure 2: Root Mean Squared Fluctuation (RMSF) comparison of the Fas DD upon Fas binding to CaM with Fas in its wildtype, V254N mutation, and C-terminal deletion mutation.

Conclusion

By combining the data from the binding free energy analysis and the conformational changes observed during the MD simulations, it was able to be determined that the V254N point mutation of Fas resulted in a less stable structure of Fas which could potentially play a role in the decreased binding affinity between Fas and CaM. Also, the C-terminal mutation resulted in a stable conformation of Fas and led to a complex that was more favorable than the wildtype case. These results provide important molecular evidence for the structural changes of Fas and CaM as a result of the formation of the CaM/Fas complex and Fas mutations. The data obtained from this study will help in the further understanding of the CaM/Fas complex and its effect on the Fas-mediated signaling pathway for the apoptosis of osteoclasts.

Acknowledgements

This work was supported in part by the NSF sponsored UAB ADVANCE program, the Mr. & Mrs. Kwok-Chong Woo Grant, and a VA Merit Award. Special thanks are extended to Di Pan for helping with the bootstrap statistical analyses, and Nathan Baker for helpful insight.

References

  1. National Osteoporosis Foundation. August 31, 2007.
  2. Seales, E. C., et al. Calmodulin is a critical regulator of osteoclastic differentiation, function, and survival. J Cell Biochem, 2006, 97(1): 45-55
  3. Ahn, E. Y., S. T. Lim, W. J. Cook, and J. M. McDonald. 2004. Calmodulin binding to the Fas death domain. Regulation by Fas activation. The Journal of biological chemistry 279:5661-5666.
  4. Suever, J., Y. Chen, J. M. McDonald, and Y. Song. 2008. Conformation and Free Energy Analyses of the Complex of Ca2+-Bound Calmodulin and the Fas Death Domain. (Biophys J , 2008, in press).

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