Author

Thomas Dunham

Date

April 2009

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

The partition system of the low copy number P1 plasmid ensures that plasmids are faithfully segregated between daughter cells using three components contained in a single par operon. The P1 par cassette includes the genes of two proteins (parA and parB) and a cis acting plasmid centromere (parS). P1 ParA is a deviant Walker A ATPase that plays an essential, but unknown role in plasmid partitioning as well as in the autorepression of its own operon. These two disparate functions are modulated by the nucleotide binding state of the ParA protein. The understanding of how nucleotide binding to ParA can affect its functional role has been limited by the absence of high-resolution structural information. X-ray crystallography studies were undertaken to address the questions centering on the nature of ParA’s different nucleotide bound states. A structure of P1 ParA in its apo state revealed that it was a dimer with an extended N-terminal α-helix. Supporting static light studies established that apo ParA is able to form a dimer in solution at physiologically relevant concentrations. The studies also revealed that the stability for the dimer state was found to be dependent upon an interaction between the N-terminal α-helix (α1) and its dimer partner. ADP binding potentiates ParA’s ability to bind DNA and function in the auto-repression of its own operon. An ADP-bound structure of P1 ParA was solved and provided the basis for a ParA-DNA-bound model that required the DNA to be significantly bent in order to align with the recognition helix of ParA’s HTH motif. A nucleotide dependent basic region revealed in an ADP-bound structure of ParA provided a mechanism to stabilize the bending of the DNA. Mutagenesis and supporting DNA binding analysis established that the basic region is important for the binding of DNA. When ParA is in its ATP-bound state it carries out a different function providing an essential role in partitioning. Negative stain electron microscopy (EM) experiments were used to demonstrate that ParA formed filaments in an ATP dependent manner. Reconstitution experiments further demonstrated that the components of the P1 par cassette were localized to the filaments suggesting that the ParA filaments are important to the segregation of the P1 plasmid. ParB has been known to modulate ParA’s ATPase activity in an unknown manner through an interaction involving its N-terminus. ParAParB fusion constructs were used to further localize both the specificity and activity features responsible for the potent interaction involving ParB’s N-terminal 28 residues. Further investigations into the source of the activity found that two arginine residues (R6 and R11) were critical for this activity possibly acting through an “arginine finger” motif mechanism. The studies on P1 ParA show that unlike other deviant Walker A ATPases that ParA likely exists as a dimer in all its states. The activity of ParA is governed by its nucleotide bound state, which locks ParA into conformations that are preferred for its disparate cellular functions – autorepression and partitioning.

Identifier

doi:10.6083/M47S7KRG

School

School of Medicine

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