Control of HslUV protease function by nucleotide binding and hydrolysis
Author(s)Yakamavich, Joseph Andrew
Massachusetts Institute of Technology. Dept. of Biology.
Robert T. Sauer.
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Many proteins act as molecular machines, using the power of nucleotide binding and hydrolysis to drive conformational changes in themselves and their target substrates. Like other AAA+ proteases, HslUV recognizes, unfolds, translocates, and degrades substrate proteins in an ATP-dependent manner. Understanding how nucleotides interact with HslU and control the activities of both HslU and HslV provides insights into the general mechanism of energy-dependent proteolysis. In order to better understand HslU-nucleotide interactions, I created a variant of HslU unable to hydrolyze ATP. HslU is composed of six identical subunits with a total of six nucleotide-binding sites. Moreover, many crystal structures show HslU with six bound nucleotides. Nevertheless, I found that HslU in solution is only able to bind 3-4 ATPs at saturation. This result rules out a model of ATP hydrolysis in which six nucleotides bind and are hydrolyzed together in a single power stroke and also suggests that many HslU crystal structures represent states that are not populated in the normal ATPase cycle. I also characterized the nucleotide requirement for various HslU activities. I found that at least two ATPs must be bound to HslU to support substrate binding and ATP hydrolysis, but showed that a single nucleotide is sufficient to support HslU-HslV binding and to stimulate HslV peptidase activity. I also found that the nucleotide state of HslU affects its affinity to HslV, weakening it when some subunits have ADP or no nucleotide bound. This effect is offset by an increase in HslU-HslV affinity during substrate degradation. This work suggests a simple model in which binding of a single ATP to HslU drives HslV binding, with further ATP binding acting to stabilize an HslU conformation that can bind protein substrate, hydrolyze ATP, and support substrate unfolding, translocation, and degradation.
Includes bibliographical references.Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2008.
DepartmentMassachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology