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Mailing Address:

   Dr. A. Clay Clark
   Associate Professor
   128 Polk Hall, CB 7622
   Department of Molecular
       & Structural Biochemistry
   NC State University
   Raleigh, NC 27695-7622

   Send Email: Clay Clark

  Lab: Rooms 339 & 341 Polk Hall
  Office: Room 339A Polk Hall

  919-515-5805 (office)
  919-515-5806 (lab)
  919-515-2047 (fax)

Research Projects

Research in my lab focuses on the broad topics of protein folding and the role of amino acid sequence identity in determining folding pathways of homologous proteins.

The first movie shows an example of the problem. Researchers learned over fifty years ago that the amino acid sequence of a protein dictates its three-dimensional structure. Since then, we have become adept at determining the amino acid sequence of a protein from the nucleotide sequence of a gene. In the example here, the white blocks might represent hydrophilic amino acids and the black blocks might represent hydrophobic amino acids. In the past several years, it has become much easier to determine the three-dimensional structure of a protein, either by x-ray crystallography or by NMR. In the example here, the structure is a cube. However, we do not understand the mechanism by which the amino acid sequence dictates three-dimensional structure, that is, we do not understand how proteins fold, and we have little information regarding the structures that form during folding.

Our research into the protein folding problem focuses on the folding and assembly of caspase proteins. An overview of caspase function in apoptosis and caspase structure is provided in a powerpoint presentation.

Caspases (cysteinyl aspartate-specific protease) are members of a family of proteases involved in apoptosis and the inflammatory response. Caspase-1 (interleukin-1b converting enzyme, ICE) and caspase-3 (CPP32, YAMA), the primary subjects of our investigations, have similar native structures, yet the amino acid sequence identity is only about 30 %. A sequence comparison is shown in the first figure. The legend for this figure can be found in MacKenzie & Clark (2007), but the colored lines correlate to the active site loops as shown in the structure of mature caspase-3 (second figure). The caspase-3 dimer contains two active sites, and each monomer consists of a large and small subunit that fold into an eight-stranded b-sheet with five a-helices that are found on the protein surface, as shown in the second figure. Caspases exist in normal cells as inactive zymogens and must be activated by proteolytic processing, as shown in the third figure above. The procaspases are single polypeptide chains that contain an amino-terminal pro-domain, which varies in size and function, a large subunit, an intersubunit linker, and a small subunit. A primary key to regulating caspase activity occurs at the level of maturation. Procaspase-3 is a stable dimer, but has very little activity, whereas procaspase-1 and the initiator procaspases are monomers that requires interactions with death scaffolds to form an active dimer. The dysregulation of caspase maturation and function may be a contributing factor to a number of human diseases - cancer, neurodegenerative diseases, and autoimmune diseases such as diabetes. Learning to selectively manipulate the levels of caspase activity may very well lead to therapeutic strategies for treatment of these diseases.

The second movie shows cells undergoing apoptosis (from Manji, G. A. & Friesen, P. D. (2001) J. Biol. Chem. 276, 16704–16710). As described above, and in the powerpoint presentation, cells are dismantled via the action of caspases and are packaged into vesicles that are cleared by macrophages.

Within this project there are three main topics:

• We are examining the folding and assembly of procaspases-3 and -1 to define the thermodynamic and kinetic folding properties of the proteins.

• We are defining the folding events that lead to active site formation in the procaspase, conformational changes that occur during maturation of caspases, and interactions in the dimer interface that affect active site formation..

  These maturation events are important because they lead to changes in the active site structure, they are thought to result in formation of the substrate binding pocket, and they result in full activity of the caspase enzyme.

  Amino acids in the dimer interface and their relation to the active site are shown in the figure above and the movie below.

• Finally, we are examining the role of the pro-domain in folding. The pro-domain of caspase-3 is 28 amino acids, and it is required for proper folding of the dimer. That is, it functions as an intramolecular chaperone. In contrast, the pro-domain of procaspase-1 is 119 amino acids. The first 92 amino acids form a CARD (caspase recruitment domain), and the remaining 27 amino acids have no known function.

 The third movie shows the relation between the dimer interface and amino acids in the active site. This movie represents several structures of wild-type caspase-3 and mutants, focused on the dimer interface. The movie shows the results of mutations at V266, which resides at the two-fold symmetry axis of the dimer, with glutamate. The glutamate residue is shown in the actual structure, an overlay with caspase-1, which naturally contains glutamate at this position, and the V266E variant modeled with the glutamate side chain in the same rotamer as for caspase-1. A space-filling model shows that there are steric clashes between the glutamate in this rotamer an Y197, also near the dimer interface and active site.

Caspase pictures were generated with Pymol. Visit the Pymol website for more information and instructions.