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Associate Professor of Biochemistry and Biophysics
Ph.D. University of Wisconsin at Madison 1995
Post-Doc. Stanford University School of Medicine 1996-1999
(Burroughs-Welcome Fund Fellow of the Life Sciences Research Foundation)
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CATALYTIC STRATEGIES OF RIBOZYMES
RNA enzymes (ribozymes) represent an emergent class of biocatalysts that engage in a diverse number of reactions ranging from protein synthesis (e.g. the ribosome) to regulation of gene expression (i.e. endonucleolytic metabolite sensors). Ribozymes are found in all kingdoms of life, and can accelerate reactions as much as 10 million-fold over uncatalyzed rates. Key questions are, "How do ribozymes accomplish rate acceleration? Are the strategies employed comparable to those of protein enzymes? How does the RNA architecture influence chemical reactivity?" To address these questions, my lab studies small ribozymes, a class of naturally occurring RNA enzymes that perform a site-specific phosphodiester bond cleavage reaction within a cognate substrate (Figure - below). Members of the family include the hammerhead, hepatitis delta virus, VS, glmS and hairpin ribozymes. Our focus is the hairpin ribozyme, because it does not require multivalent ions to participate directly in the chemical steps and its reaction is readily reversible.
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Schematic Depictions of the Hairpin Ribozyme Mechanism of Action. (A) ‘B’ represents an unidentified specific base catalyst, such as water. A38 serves as a general acid. (B) Alternatively, A38 may serve as a proton shuttle for an unidentified specific acid ‘AH’. From Fedor & Williamson (2005) Nat. Rev. Mol. Cell Biol. 19, 27-37.
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Research in my lab involves X-ray crystallographic, kinetic and synthetic organic methods. Recently we determined
the crystal structure of a minimal all-RNA variant of the hairpin ribozyme and refined its structure to 2.15 Å resolution (Figure – Global Structure). The crystallization construct was produced entirely by solid phase chemical synthesis, demonstrating the feasibility of replacing any functional group for structure-function analyses (comparable to site directed mutagenesis of a protein). This study revealed the basis for the metal dependence of ‘gain-of-function’ mutations U39C and U39(propyl),
and showed how these substitutions shift the equilibrium conformation of U37 in the S-turn from an exposed form, observed in the native U39 structure, to a sequestered form in which U37 resides in close proximity to active site residue G+1. In another study, replacement of the active site base, G8, with various analogues (e.g. inosine, diaminopurine, aminopurine, adenine and uracil) changed the local in-line geometry, demonstrating the importance of individual functional groups in maintaining the catalytic configuration for phosphoryl transfer (Figure – Active Site). Perhaps our most exciting recent finding was the presence of ordered water molecules
in the hairpin ribozyme active site. We are currently investigating the significance of these waters through synthesis of novel nucleotide bases that influence the mode and location of water coordination. Water has been invoked in the hairpin ribozyme mechanism of action (above) and may represent a general strategy to diversify the functional repertoire of catalytic RNA.
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STRUCTURAL ANALYSIS OF RNA EDITING COMPLEXES AND APOBEC RELATED PROTEINS (ARPS)
APOBEC-1 is the core editing enzyme of a multiprotein complex
 that C-to-U 'edits' a specific site within ApoB mRNA transcripts. Editing produces a premature stop codon that leads to a truncated form of the serum lipoprotein apoB. The absence of apoB mRNA editing produces full-length apoB, which is associated with LDL atherosclerotic risk in humans. Recently, several ARPs were identified in the human genome including the activation induced deaminase (AID) and APOBEC-3G (hA3G). AID is essential for somatic hypermutation and class switch recombination of Ig genes, whereas hA3G restricts HIV-1 infectivity in non-permissive cells. Although these proteins do not appear to edit RNA, each exhibits dC-to-dU deaminase activity on DNA. Recent studies indicate APOBEC-1 also mutates DNA suggesting a novel mechanism for ARP-mediated genome mutagenesis leading to neoplastic transformation.
 My lab has uses comparative modeling, X-ray crystallography and biochemical analysis to study the basis for ARP substrate specificity and the role of auxiliary factors in enzyme regulation. Recently we solved the structure of a yeast cytidine deaminase capable of editing reporter apoB mRNA in yeast cells (Figure – Cytidine Deaminase). This structure provided a basis to construct three-dimensional molecular models of APOBEC-1 and AID, which are being used as a basis to probe structure-function relationships. Overall, this work is relevant in the identification of novel mechanisms of cancer, treating atherosclerotic disease and enhancing innate antiviral defense mechanisms. We work in close collaboration with the labs of
Profs. Harold C. Smith
and
Andrea Bottaro.
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Visit my Lab Page
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Spitale RC, Heller MG, Pelly AJ, Wedekind JE (2007) Efficient Syntheses of 5'-Deoxy-5'-fluoroguanosine and -inosine. J Org Chem,
Lehmann DM, Galloway CA, MacElrevey C, Sowden MP, Wedekind JE, Smith HC (2007) Functional characterization of APOBEC-1 complementation factor phosphorylation sites. Biochim Biophys Acta, 1773:408-18
Macelrevey C, Spitale RC, Krucinska J, Wedekind JE (2007) A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme. Acta Crystallogr D Biol Crystallogr, 63:812-25
Torelli AT, Krucinska J, Wedekind JE (2007) A comparison of vanadate to a 2'-5' linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization. Rna, 13:1052-70
Bennett RP, Diner E, Sowden MP, Lees JA, Wedekind JE, Smith HC (2006) APOBEC-1 and AID are nucleo-cytoplasmic trafficking proteins but APOBEC3G cannot traffic. Biochem Biophys Res Commun, 350:214-9
Chaves FA, Richards KA, Torelli A, Wedekind J, Sant AJ (2006) Peptide-binding motifs for the I-Ad MHC class II molecule: alternate pH-dependent binding behavior. Biochemistry, 45:6426-33
Ichikawa HT, Sowden MP, Torelli AT, Bachl J, Huang P, Dance GS, Marr SH, Robert J, Wedekind JE, Smith HC, Bottaro A (2006) Structural phylogenetic analysis of activation-induced deaminase function. J Immunol, 177:355-61
Salter J, Krucinska J, Alam S, Grum-Tokars V, Wedekind JE (2006) Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer. Biochemistry, 45:686-700
Wedekind JE, Gillilan R, Janda A, Krucinska J, Salter JD, Bennett RP, Raina J, Smith HC (2006) Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits. J Biol Chem, 281:38122-6
Ghosh AK, Sridhar PR, Leshchenko S, Hussain AK, Li J, Kovalevsky AY, Walters DE, Wedekind JE, Grum-Tokars V, Das D, Koh Y, Maeda K, Gatanaga H, Weber IT, Mitsuya H (2006) Structure-Based Design of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance. J Med Chem, 49:5252-5261
Xie K, Song SC, Spitalnik SL, Wedekind JE (2005) Crystallographic analysis of the NNA7 Fab and proposal for the mode of human blood-group recognition. Acta Crystallogr D Biol Crystallogr, 61:1386-94
Gruswitz F, Frishman M, Goldstein BM, Wedekind JE (2005) Coupling of MBP fusion protein cleavage with sparse matrix crystallization screens to overcome problematic protein solubility. Biotechniques, 39:476, 478, 480
Alam S, Grum-Tokars V, Krucinska J, Kundracik ML, Wedekind JE (2005) Conformational Heterogeneity at Position U37 of an All-RNA Hairpin Ribozyme with Implications for Metal Binding and the Catalytic Structure of the S-Turn(,). Biochemistry, 44:14396-408
Jamburuthugoda VK, Guo D, Wedekind JE, Kim B (2005) Kinetic evidence for interaction of human immunodeficiency virus type 1 reverse transcriptase with the 3'-OH of the incoming dTTP substrate. Biochemistry, 44:10635-43
Smith HC, Wedekind JE, Xie K, Sowden MP (2005) Mammalian C to U Editing. Topics in Current Genetics: "Fine-Tuning of RNA Functions by Modification and Editing" H. Grosjean (Ed.) Springer-Verlag, Berlin, 1610-2096
MacElrevey C, Wedekind JE (2005) Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design. Acta Crystallogr F Biol Crystallogr, 61:1049-53
Goodman JL, Wang S, Alam S, Ruzicka FJ, Frey PA, Wedekind JE (2004) Ornithine cyclodeaminase: structure, mechanism of action, and implications for the mu-crystallin family. Biochemistry, 43:13883-91
Xie K, Sowden MP, Dance GS, Torelli AT, Smith HC, Wedekind JE (2004) The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1. Proc Natl Acad Sci U S A, 101:8114-19
Alam S, Wang SC, Ruzicka FJ, Frey PA, Wedekind JE (2004) Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida. Acta Crystallogr D Biol Crystallogr, 60:941-4
Smith HC, Bottaro A, Sowden MP, Wedekind JE (2004) Activation induced deaminase: the importance of being specific. Trends Genet, 20:224-7
Song SC, Xie K, Czerwinski M, Spitalnik SL, Wedekind JE (2004) Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen. Acta Crystallogr D Biol Crystallogr, 60:788-91
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Graduate students in my laboratory work toward a Ph.D. degree in the following program[s]:
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Ph.D. in Biochemistry
Ph.D. in Biophysics
Ph.D. in Chemistry
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Ph.D. candidates in my laboratory may also be affiliated with these programs:
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click here to learn more and to apply to graduate school |
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E-Mail: Joseph_Wedekind@urmc.rochester.edu
Joseph E. Wedekind
Department of Biochemistry and Biophysics
University of Rochester School of Medicine and Dentistry
601 Elmwood Ave, Box 712
Rochester, New York 14642
Office: Medical Center 3-8537
Telephone: (585) 273-4516; Fax: (585) 275-6007
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