This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Desulfitobacterium dehalogenans is an anaerobic microbe that can harvest energy by coupling reductive dehalogenation of chloroaromatic substrates to an electron transport chain in a process called dehalorespiration (1, 2). The cpr gene cluster is an eight-gene cluster that is induced when anaerobes sense chlorinated aromatic compounds in their environment, many of which are environmental pollutants (3). The reductive dehalogenase (CprA), containing vitamin B12 and FeS clusters, has been shown to catalyze the dehalogenation of hydroxyl-PCB and other chloroaromatics (4). Expression of CprA and other components of the cpr gene cluster was proposed to be controlled by CprK based on its sequence similarity to FNR and FixK, global regulators of anaerobic metabolism (3). CprK also shares sequence similarity with cAMP receptor protein, CRP, leading to the suggestion that CprK might be a member of the CRP-FNR superfamily of transcriptional regulators. My collaborator Steve Ragsdale and his laboratory focus not only on elucidating the catalytic mechanism of the reductive dehalogenase (CprA) from D. dehalogenans, but also on elucidating the mechanism by which this microbe senses toxic chloroaromatics and regulates dehalorespiration. Stelian Pop, a graduate student in the Ragsdale lab, has characterized CprK and demonstrated that the protein, upon binding a chlorinated aromatic, activates transcription of the cpr gene cluster, the dehalorespiration operon. He has also recently discovered that this protein only binds DNA under anaerobic conditions and is redox regulated, active only when reduced (unpublished results, Pop & Ragsdale). A well known member of the CRP-FNR superfamily of transcriptional regulators is CRP itself. The allosteric regulation of this protein is well studied and the role of the effector-mediated CRP conformation changes central to its function as a transcription factor are well understood (5). CprK, on the other hand, utilizes the unique effector 3-chloro-4-hydroxyphenyacetate (CHPA), an effector quite unlike cAMP. Therefore, a structural study of the effector-mediated allosteric control of the conformation and activity of CprK would allow an evaluation of the structural basis of a unique effector-effector domain interaction, as well as the CprK-cpr promoter region. Additionally, the structural studies proposed would be the first study of a transcriptional regulatory protein that responds to toxic PCBs. Finally, the newly discovered redox regulation of this protein will also be explored by obtaining structures of both active and inactive conformers, offering insight into yet another layer of regulation of CprK. To complement the active, ongoing functional studies in the Ragsdale lab, structural studies of CprK, with the following long-term goals, are proposed. We plan to elucidate the structural details of the redox regulation of CprK. X-ray crystallographic techniques and methodologies will be employed in order to determine the following structures, The inactive conformation of CprK under aerobic conditions and, The active conformation of CprK under anaerobic conditions.Comparison of the two structures will provide valuable insight into how CprK is regulated by its redox state. We plan to elucidate the structural details of the effector-mediated allosteric control of the conformation and activity of CprK. Both the interaction between CprK and the cpr promoter region and between effector and the effector domain are to be explored. X-ray crystallographic techniques and methodologies will be employed to evaluate the structural basis of interactions in both regions. Specifically, the following structures will be determined, CprK with effector bound and, CprK with effector and DNA bound. During Summer 2004, Anthony Krueger crystallized the active form of CprK under anaerobic conditions as a participant in the University of Nebraska-Lincoln, Redox Biology Center Summer Research Program (Dr. Ragsdale and Dr. Ryter, co-mentors). These crystals were subsequently characterized at the Structural Biology Core Facility at UNL. The diffraction pattern revealed the crystals were indeed protein and diffracted to a resolution of 4 . The crystals were subsequently repeated and their diffraction limit improved to 3 by Ben Biehl, a NWU 2004 graduate and lab technician in the Ragsdale lab. During Fall 2004, Mr. Biehl also successfully crystallized CprK with CHPA bound, and it is hoped that further refinement of the crystallization conditions will result in diffraction quality crystals. The CprK crystals have been sent to Stanford Synchrotron Radiation Laboratory for native data collection. Mr. Biehl is currently working on making a Se-Met derivative of the protein to crystallize so that MAD phasing may be utilized in structure determination.References1. Utkin, I., C. Woese, et. al. (1994). Int J Syst Bacteriol 44(4): 612-9.2. Wiegel, J., X. Zhang, et. al. (1999). Appl Environ Microbiol 65(4): 2217-21.3. Smidt, H., M. van Leest, et. al. (2000). J Bacteriol 182(20): 5683-91.4. Krasotkina, J., T. Walters, et. al. (2001). J Biol Chem 276(44): 40991-7.5. Harman, J.G. (2001). Biochim Biophys Acta 1547(1): 1-17.6. www.hamptonresearch.com/support/pdf101/CG101SDC.pdf

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Exploratory Grants (P20)
Project #
5P20RR016469-07
Application #
7627625
Study Section
Special Emphasis Panel (ZRR1-RI-7 (01))
Project Start
2007-05-01
Project End
2008-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
7
Fiscal Year
2007
Total Cost
$42,933
Indirect Cost
Name
University of Nebraska Medical Center
Department
Genetics
Type
Schools of Medicine
DUNS #
168559177
City
Omaha
State
NE
Country
United States
Zip Code
68198
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