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
• Altering pH of the system by changing the protonated states of specific residues
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 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.
Figure 3. The residues highlighted in blue are changed to protonated (uncharged) state to simulate pH=5.5-‐6.0 condiAon; the residues highlighted in blue and orange are changed to protonated state to simulate pH=4.3-‐4.6 condiAon.
Fig 1a. Capsid filled with RNA
Fig 1b. Empty capsid without RNA
(Prepared by mixing capsid
(Prepared from pure capsid protein,
protein with RNA )
and lowered pH to around 4.5)
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.
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) 10,000 STEPS
• 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 Figure 2. The dashed curve corresponds to the VDW and solvaAon interacAon between two carbon atoms. The step funcAon is its DMD discreAzed approximaAon.
• Comparison between equilibrium structure in solution and the crystal (virion) structure of the CCMV capsid protein dimer Figure 4. Energy curve versus number of steps (Ame). Each step in the graph represents 10 steps in the simulaAon. 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. Clamp structure Clamp structure Figure 5. 3D ribbon crystal (virion) structure (leC) and equilibrium soluAon structure (right) of CCMV capsid protein dimer in pH=4.3-‐4.6 aCer 50,000 simulaAon steps. Blue sequences represent the condiAon 30th-‐45th amino acid residues of the protein N terminus and red sequences represent the 180th-‐190th amino acid residues of the C terminus. Earlier research 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', two-‐fold related subunit, clamps the interpenetraAng C terminal arm of the other one between itself and the invaded β-‐barrel. 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 Figure 5. Comparison between the virion structure (light brown) and the equilibrium soluAon structure in pH=5.5-‐6.0 (light blue) (leC), and between the virion structure (light brown) and the equilibrated structure in pH=4.3-‐4.6 (pink) (right). Blue sequences represent the 30th-‐45th amino acid residues of the N th-‐190th amino terminus a nd r ed s equences r epresent t he 1 80 acid residues of the C terminus.
Contact Xu, Tianchuan Nanjing University Email:firstname.lastname@example.org
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. :neutral pH(pH=7.0) :low pH (pH=4.3-‐4.6) Figure 6. Dihedral angle between two β-‐sheet 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 Dimer structure in capsid from a new trajectory aCer equilibraAon at the given pH. The data used to make a plane ﬁt are from the alpha carbons of each residue in the β-‐sheet secondary structure nearby the N and C terminal. The dihedral angles under pH=7.0 and pH=4.3-‐4.6 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=4.3-‐4.6 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=4.3-‐4.6 and virion structure is 1.155 angstroms, again consistent with the results above.
• 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.
Future Plans • 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
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. Yung-‐ya Lin, Crystal Valdez, and Xinkai Fu.
References 1. 2. 3. 4.
Rees F. Garmann, Mauricio Comas-‐Garcia, Ajaykumar Gopal, Charles M. Knobler, and William M. Gelbart. (2013). The Assembly Pathway of an Icosahedral Single-‐Stranded RNA Virus Depends on the Strength of Inter-‐Subunit AtracAon. J. Mol. Biol., in press. Feng Ding, Douglas Tsao, Huifen Nie, and Nikolay V. Dokholyan. (2008). Folding with All-‐Atom Discrete Molecular Dynamics. Structure 16, 1010–1018. 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. 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 X-‐ray crystallography and cryo-‐electron microscopy. Structure 3, 63-‐78.