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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[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.

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[3].  



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

30,000  STEPS

50,000  STEPS

100,000  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.[2]

  • 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[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',  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  

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.  

Research Poster of Xu Tianchuan Nanjing University