Structural studies of Sindbis virus capsid protein mutants

Sukyeong Lee, Purdue University

Abstract

Alphaviruses are icosahedral, positive-stranded, enveloped RNA viruses. Crystals of Sindbis, Semliki Forest, and Ross River viruses had been obtained previously but diffracted only to 30 A resolution. However the crystal structures of the C-terminal domains of SINV and SFV capsid proteins had been solved. In this thesis, are presented the structures of two Sindbis virus capsid protein mutants expressed in E. coli. The structure of the mutant SCP(S215A, 106-266) was studied to analyze the catalytic properties of the protein, which cleaves itself from the structural polyprotein. SCP(S215A, 106-266) has two extra amino acids after the C-terminal cleavage site and the catalytic Ser215 was mutated to alanine to prevent cleavage. Therefore this mutant represents the substrate-enzyme complex of this autocatalytic proteinase. The two molecules in the crystal unit cell showed different substrate binding states. This mutant structure was compared with wild-type SCP, other viral proteinases, and cellular proteinases. A structural basis is discussed for the efficiencies of different nucleophiles and active site geometries. This mutant structure also showed additional N-terminal amino acids bound into a hydrophobic pocket on the surface of a neighboring molecule. This and other biochemical and genetic studies lead to the suggestion that the mode of binding used by the N-terminal arm is analogous to the binding of the glycoprotein E2 to the capsid protein. In addition, a "switch" mechanism was proposed for the interaction of glycoprotein with capsid protein based on the structural comparison of the hydrophobic pockets of mutant and wild-type SCP (which has the N-terminal arm bound to the pocket) with wild-type SFCP (which does not have a bound N-terminal arm). To test the proposed mechanism, SCP(114-264), which is missing the N-terminal arm, was crystallized to examine the pocket conformation when the pocket is empty. However, the pocket was occupied by dioxane molecules from the crystallization solution. The pocket conformation was the same of that when the pocket was occupied by the N-terminal arm. In the switch mechanism, the structural change occurs between the empty pocket, represented by cytoplasmic cores, and the occupied pocket, represented by cores in the virus. However genetic studies conducted by Katherine Owen and Richard Kuhn showed that binding of the N-terminal arm to the capsid protein has a role in core assembly. Thus, if the N-terminal arm is already bound in the cytoplasmic cores, the glycoprotein replaces the N-terminal arm. This would make the observation of an empty pocket as found in SFCP not necessarily relevant. Further attempts to verify the interaction of glycoprotein E2/capsid protein were made. (i) A SFCP mutant in which the N-terminal arm residues are mutated to the glycoprotein E2 sequence was expressed; (ii) Core purification and crystallization was tried to determine the structures of cores and E2-like peptide/core complex.

Degree

Ph.D.

Advisors

Rossman, Purdue University.

Subject Area

Biophysics|Microbiology|Biochemistry

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