Oligomerization of the SPP1 scaffolding protein.
Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His(6)) and purified. The protein is an alpha-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His(6) is structurally a dimer of dimers. Incubation at temperatures above 37 degrees C correlated with a reduction of its alpha-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 degrees C. Refolding of gp11-His(6) thermally denatured at 65 degrees C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (approximately 90%) and a smaller fraction of 2.4 S dimers (approximately 10%). This population of gp11-His(6) was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His(6) showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His(6).
Poh
Siew Lay
SL
Unité de Virologie Moléculaire et Structurale, UMR CNRS 2472, UMR INRA 1157 and IFR 115, Bât. 14B, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
el Khadali
Fatima
F
Berrier
Catherine
C
Lurz
Rudi
R
Melki
Ronald
R
Tavares
Paulo
P
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000145:classification; D002942:Circular Dichroism; D019281:Dimerization; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D014764:Viral Proteins; D006639:Histidine