Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids.
Sesbania mosaic virus (SeMV) capsids are stabilized by RNA-protein, protein-protein and calcium-mediated protein-protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS). The CBS environment is found to be identical even in the recombinant CP-NDelta65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146N-D149N mutation in CP-NDelta65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the T=3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the beta-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM-RNA interactions that lead to the formation of the beta-annulus, which is essential for T=3 capsid assembly.
Satheshkumar
P S
PS
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Lokesh
G L
GL
Sangita
V
V
Saravanan
V
V
Vijay
C S
CS
Murthy
M R N
MR
Savithri
H S
HS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D018360:Crystallography, X-Ray; D004279:DNA, Viral / Q000235:genetics; D007887:Fabaceae / Q000821:virology; D008854:Microscopy, Electron; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000737:chemistry* / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014357:Trypsin; D019065:Virus Assembly
D036022:Capsid Proteins; D004279:DNA, Viral; D021122:Protein Subunits; D011994:Recombinant Proteins; D014357:Trypsin; D002118:Calcium