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• Comparison between equilibrium structure in solution and the crystal (virion) structure of the CCMV capsid protein dimer From the graph above, the CCMV protein dimer achieves its equilibrium state in 30,000 steps (1.5 ns) of simulaAon in all pH condiAons. No big energy change occurs aCer the first 5,000 steps (0.25 ns), which indicates the dimer does not undergo a significant transformaAon in soluAon environment. Earlier research [4] suggests that the most important interacAon between CCMV capsid protein dimer is a special structure referred to as a “clamp”: the N terminal arm of the 'invaded', twofold related subunit, clamps the interpenetraAng C terminal arm of the other one between itself and the invaded βbarrel. Figure 5 shows that this “clamp structure” of CCMV capsid protein dimer remains the same in its equilibrated state in soluAon. • Comparison between equilibrium structures in solution at different pHs Results Fig. 5 shows the conformational change of one monomer in the dimer by maximizing the overlap of the other. It indicates that under normal pH conditions, the structure of a single monomer of CCMV protein dimer differs a lot from its virion structure. However, when lowering pH to 4.3-4.6, the structure undergoes a ‘return transition’ and shows good resemblance with the virion crystal structure. The data used to make a plane fit are from the alpha carbons of each residue in the βsheet secondary structure nearby the N and C terminal. The dihedral angles under pH=7.0 and pH=4.34.6 are around 99 degrees and 90 degrees respecAvely. The dihedral angle of the beta sheet in the virion structure, calculated by the same method, is about 85 degrees. This shows the soluAon structure at pH=4.34.6 is closer to that in the crystal structure than is that at pH=7.0. In addiAon, the RMSD value between the structure at pH=7.0 and in the virion is 1.236 angstroms, while the RMSD between the pH=4.34.6 and virion structure is 1.155 angstroms, again consistent with the results above. ConformaCon of CCMV Capsid Protein Dimer, in the intact virion and in soluCon at different pHs Tianchuan Xu, Nanjing University; Charles M. Knobler and William M. Gelbart, UCLA Xu, Tianchuan Nanjing University Email:[email protected] Contact 1. Rees F. Garmann, Mauricio ComasGarcia, Ajaykumar Gopal, Charles M. Knobler, and William M. Gelbart. (2013). The Assembly Pathway of an Icosahedral SingleStranded RNA Virus Depends on the Strength of InterSubunit AtracAon. J. Mol. Biol., in press. 2. Feng Ding, Douglas Tsao, Huifen Nie, and Nikolay V. Dokholyan. (2008). Folding with AllAtom Discrete Molecular Dynamics. Structure 16, 1010–1018. 3. Florence Tama and Charles L. Brooks III. (2002). The Mechanism and Pathway of pH Induced Swelling in Cowpea ChloroAc Motle Virus. J. Mol. Biol. 318, 733–747. 4. Jeffrey A. Speir, Sanjeev Munshi, Guoji Wang, Timothy S. Baker and John E. Johnson. (1995). Structures of the naAve and swollen forms of cowpea chloroAc motle virus determined by Xray crystallography and cryoelectron microscopy. Structure 3, 6378. References Cowpea chlorotic mottle virus (CCMV), an icosahedral (T=3) RNA plant virus, has been studied for 50 years, ever since it was the first spherical virus to be reconstituted in vitro from purified components. Recent research [1] in our group has established that with the help of RNA, CCMV capsid protein dimers could self assemble at neutral pH. But more interesting is, CCMV capsid protein dimers could self assemble an empty capsid without RNA at low pH (pH around 4.5) with high ionic strength. While the structure of the CCMV protein dimer in the virion has been determined from crystallography and cryo-electron microscopy, its conformation in solution is not known. To get a better understanding of the mechanism of its self-assembly into virions, we set out to learn about the structure of the dimer from molecular dynamics simulation and – in particular – to learn how its conformation changes with pH. IntroducCon Our project can be divided into two steps: 1. Obtain the equilibrium structure of CCMV dimer in solution and compare with its structure in the virion; 2. Determine how conformation changes as a function of pH. • Use the method of discrete molecular dynamics (DMD) simulation and related software to run trajectories for a sufficient number of steps to achieve equilibration (each step = 50 femtoseconds) • Use all-atom and implicit solvent models to treat the protein dimer and water molecules, respectively • Use step-well potential functions to replace continuous screened coulomb, Leonard-Jones, and other effective interactions between atom pairs Methods • The crucial ‘clamp structure’ stabilizing the capsid protein dimer – determined from high-resolution studies of the virion – remains intact upon equilibrating the dimer in solution. • The solution structure of CCMV protein dimer at pH=4.3-4.6 is closer to that of the virion structure than is the pH=7.0 dimer. This conclusion is consistent with recent experimental results from our lab showing that self-assembly of capsids from dimers is facilitated by lowering of the pH from neutral to values in the range of 4.5, both in the presence and absence of RNA. Conclusions This work is supported by the UCLA CSST Program. I appreciate the great help from Prof. William M. Gelbart, Prof. Charles M. Knobler, Prof. Anastassia N. Alexandrova, Prof. Yungya Lin, Crystal Valdez, and Xinkai Fu. Acknowledgement • Altering pH of the system by changing the protonated states of specific residues 10,000 STEPS 30,000 STEPS 50,000 STEPS 100,000 STEPS Figure 2. The dashed curve corresponds to the VDW and solvaAon interacAon between two carbon atoms. The step funcAon is its DMD discreAzed approximaAon. [2] Figure 3. The residues highlighted in blue are changed to protonated (uncharged) state to simulate pH=5.56.0 condiAon; the residues highlighted in blue and orange are changed to protonated state to simulate pH=4.34.6 condiAon [3] . Figure 4. Energy curve versus number of steps (Ame). Each step in the graph represents 10 steps in the simulaAon. Figure 5. 3D ribbon crystal (virion) structure (leC) and equilibrium soluAon structure (right) of CCMV capsid protein dimer in pH=4.34.6 condiAon aCer 50,000 simulaAon steps. Blue sequences represent the 30 th 45 th amino acid residues of the protein N terminus and red sequences represent the 180 th 190 th amino acid residues of the C terminus. Figure 6. Dihedral angle between two βsheet structures of different monomers in one dimer. Each point is the average of 10 calculaAons of dihedral angle corresponding to 10 dimer configuraAons chosen randomly from a new trajectory aCer equilibraAon at the given pH. Figure 5. Comparison between the virion structure (light brown) and the equilibrium soluAon structure in pH=5.56.0 (light blue) (leC), and between the virion structure (light brown) and the equilibrated structure in pH=4.34.6 (pink) (right). Blue sequences represent the 30 th 45 th amino acid residues of the N terminus and red sequences represent the 180 th 190 th amino acid residues of the C terminus. • Improving the method of simulating changes under the pH conditions of biomolecular system in solution • Altering the linear algorithm of potential calculation by parallelization of the code • Taking into explicit account the RNA and its role in the self-assembly process, as well as important metal (e.g., divalent) counterions and N terminus • Calculating the RMSD value for each monomer in order to separate the influence of relative motion of two monomers from conformaAonal changes within the individual monomer subunits Future Plans Fig 1a. Capsid filled with RNA (Prepared by mixing capsid protein with RNA ) Clamp structure Clamp structure Dimer structure in capsid :neutral pH(pH=7.0) :low pH (pH=4.34.6) Fig 1b. Empty capsid without RNA (Prepared from pure capsid protein, and lowered pH to around 4.5)
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