Research:

Poly(A) Polymerase

Polyadenylation is an essential step in mRNA synthesis, and defects in this process profoundly interfere with normal cell function. In humans such defects have been shown to cause thalassemias, lysozomal storage disorders, and a form of muscular dystrophy. The poly(A) polymerase (PAP) responsible for post-transcriptional mRNA elongation in eukaryotes does not require a template strand. As such, this enzyme is fundamentally different from the vast majority of other polymerases that have been structurally characterized. To form a poly(A) tail, PAP has evolved a mechanism that allows its amino acids to encode specificity for RNA as a primer and adenosine as the base of the incoming nucleotide triphosphate.

Our crystal structure of Poly(A) polymerase (PAP) shows that the enzyme is organized into three domains of 150-200 amino acids each. The N-terminal domain belongs to the nucleotidyl transferase (NT) superfamily of enzymes, and contains the hallmark triad of carboxylic amino acids. These carboxylic amino acids participate in the metal-coordinated binding of the ATP phosphate groups, and are responsible for positioning the nucleophilic 3'-OH of the primer to facilitate the nucleotidyl transferase reaction. Mutagenesis studies have implicated residues within the middle domain in differentiating between RNA and DNA primers, and in binding a variety of factors which regulate polyadenylation. The C-terminal region of PAP is responsible for holding the single stranded poly(A) primer in place. Template-directed polymerases use a "thumb domain" to hold onto their substrate via the template strand. Since PAP does not utilize a template, the thumb domain is missing, and the responsibility for holding the substrate has been largely shifted to the C-terminal domain. In our crystal structure, we see two molecules of 3'deoxy-ATP bound to the enzyme. One (shown in red) binds the expected position of the incoming nucleotide. The other (yellow) binds a site that is biochemically consistent with that expected for the 3' end of the mRNA substrate. Curiously, we do not see any clear interactions between the incoming nucleotide and the base of the incoming ATP, suggesting that the protein and/or nucleotide must adopt some other conformation during the part of the reaction in which the base is recognized by the enzyme.

Our current research in this area is focused on understanding the structural basis for PAP processivity. In addition to PAP itself, we are also pursuing crystallization trials involving a variety of proteins which regulate PAP. In the cell, PAP exists as part of the multi-component cleavage/polyadenylation macromolecular assembly. Components of this assembly (most notably Fip1 in the yeast system) regulate both activity and processivity.

Edema Factor/Calmodulin Complex

Certain pathogenic bacteria have evolved exotoxins which severely disrupt the internal cAMP levels of infected cells. Bacillus anthracis is one such bacteria, and the adenylyl cyclase exotoxin from anthrax is known as edema factor. Presumably to overcome the problem of unregulated cAMP production within the bacteria, the edema factor enzyme has evolved to become active only upon binding calmodulin (which is endogenous to the host but not the bacteria). During the course of anthrax infection, edema factor acts in concert with the lethal factor exotoxin of Bacillus anthracis, which proteolytically inactivates ERK kinase (MAP-kinase kinase). The unregulated levels of cAMP associated with anthrax are thought to be involved in both cutaneous and systemic anthrax infection.

Edema factor is between 100 and 1000 fold more active than mammalian adenylyl cyclases. Unlike mammalian cyclases, which are a membrane proteins with two homologous catalytic core domains, edema factor is a soluble protein, and shows no apparent sequence or structural homology to the cyclases from higher eukaryotes. We have solved crystal structures of both the catalytic domain of edema factor alone and the catalytic domain in complex with calmodulin.

Crystal structures of edema factor in its inactive state (left) and its calmodulin-bound active state (right). The enzymatic core of edema factor (green) binds a metal ion (purple) at the active site of both structures. In the inactive state, edema factor's helical domain (yellow) is tightly associated with the tuquoise and magenta loops of the enzyme. Calmodulin (red) activates edema factor by inserting itself between the turquoise and yellow segments, thus causing a complete rearrangement of the purple loop. In its active-state conformation, the purple loop stabilizes the orange loop, which in turn positions the substrate for catalysis.

For more details, please see the publications below.

Balbo, Paul B; Meinke, Gretchen; Bohm, Andrew. (2005). “Kinetic Studies of Yeast PolyA Polymerase Indicate an Induced Fit Mechanism for Nucleotide Specificity.” Biochemistry 44(21): 7777-7786

Swift, Steven; Leger, Andrew J; Talavera, Joyce; Zhang, Lei; Bohm, Andrew; Kuliopulos, Athan. (2006). “Role of the PAR1 Receptor 8th Helix in Signaling: The 7-8-1 Receptor Activation Mechanism.” Journal of Biological Chemistry 281(7): 4109-4116

Meinke, Gretchen; Bullock, Peter A; Bohm, Andrew. (2006). “Crystal structure of the SV40 large T-antigen origin-binding domain.” Journal of Virology 80(9): 4304-4312

Balbo, P; Meinke, G; Bohm, A. (2005) Kinetic Studies of Yeast Poly(A) Polymerase Indicate an Induced Fit Mechanism for Nucleotide Selectivity. Biochemistry, in press.

Zhelkovsky, A, Helmling, S., Bohm, A., Moore, C. (2004) Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP. RNA 10(4): 558-64.

Shen, Y., Guo, Q., Zhukovskaya, N.L., Drum, C.L., Bohm, A., Tang, W.-J. Structure of anthrax edema factor-calmodulin sensitive adenosine-(alpha, beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site. (2004) Biochem Biophys Res Commun. 317(2) 309-14.

Obin, M., Leyy, B.Y., Meinke, G., Bohm, A., Lee, R.H., Gaudet, R., Hopp, J.A., Arshavsky, V.A., Willardson, B.A., Taylor, A. (2002) Ubiquination of the Trasducin beta gamma Subunit Complex. Journal of Biological Chemistry 277, 44566-44575.

Drum, C.L., Yan, S.-Z., Bard, J., Shen, Y.-Q., Lu, D., Soelaiman, S., Grabarek, Z., Bohm, A., Tang, W.-J. (2002) Structural basis for the activation of the anthrax adenylyl cyclase exotoxin by calmodulin. Nature 415, 396-402.

Drum, C.L., Shen, Y., Rice, P.A., Bohm, A., Tang, W.-T. Crystallization and Preliminary X-ray Study of the Edema Factor Exotoxin Adenylyl Cyclase Domain from Bacillus Anthracis in the Presence of its activator, Calmodulin. (2001) Acta Crystallographica D57, 1881-1884.

Skiba N.P., Martemyanov, K.A., Elfeinbein, A., Hopp, J.A., Bohm, A., Simonds, W.F., and Arshavsky, V.Y. (2001) GS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex. Journal of Biological Chemistry 276:37365-72.

Drum, C.L., Yan, S.-Z., Sarac, R., Mabuchi, Y., Beckingham, K., Bohm, A., Grabarek, Z., Tang, W.-J. (2000) An Extended Conformation of Calmodulin Induces Interactions between the Structural Domains of Adenylyl Cyclase from Bacillus anthracis to Promote Catalysis. Journal of Biological Chemistry 275, 36334-340.

Bard J, Zhelkovsky AM, Helmling S, Earnest TN, Moore CL, Bohm A. (2000) Structure of yeast Poly(A) polymerase alone and in complex with 3'-dATP. Science 289, 1346-1349.

Balbo, PB., Meinke, G. and Bohm, A. (2005) Kinetic studies of yeast poly(A) polymerase indicate an induced fit mechanism for nucleotide specificity. Biochemistry. 44(21): 7777-86.

Lab Members

	Paul Balbo, Ph.D.  (Research Associate) Paul.Balbo@tufts.edu
	Gretchen Meinke, Ph.D. (Research Associate) Gretchen.Meinke@tufts.edu

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