John R. Murphy, Ph.D.
B.A. University of Connecticut
Ph.D. University of Connecticut School of Medicine
Our research is focused in four areas: structure and genetic analysis of the diphtheria toxin repressor (DtxR), molecular mechanisms of diphtheria toxin catalytic domain delivery into the eukaryotic cell cytosol, diphtheria toxin-based cytokine fusion proteins, and structure function analysis of interleukin 7. The regulation of diphtheria tox gene expression is controlled by DtxR, an iron-activated repressor which also controls the expression of a constellation of iron-sensitive genes. The dtxR gene has been cloned and DtxR has been overexpressed in recombinant Escherichia coli. The X-ray crystal structure of both the apo- and metal ion activated forms of DtxR, and of the ternary complex between DtxR/Ni2+/tox operator DNA have been partially solved. We have developed a positive genetic selection system for the direct cloning of both dtxR alleles and DtxR target operator sequences. Using this system, we have isolated the first positive mutants of DtxR. Since DtxR and its homologues control the expression of virulence factors in many important Gram positive pathogens, the positive dominant mutants suggest that DtxR may be a target for the development of a new class of “antimicrobial” that would phenotypically convert pathogenic strains to their non-pathogenic counterparts.
We are also focused on the structure/function analysis of the diphtheria toxin molecule. Diphtheria toxin contains at least three functional domains: ADP-ribosyltransferase, membrane translocation domain, and a eukaryotic cell receptor binding domain. Biochemical genetic analysis has shown that each domain must function in an ordered and sequential fashion for the toxin molecule to bind to its receptor on the surface of a sensitive eukaryotic cell, be internalized, and facilitate the delivery of the ADP-ribosyltransferase to the cytosol. To further probe the structural requirements for ADP-ribosyltransferase entry into the cytosol, we have designed a family of “new” toxins by receptor binding domain substitution. The first of these fusion toxins has been approved by the Food and Drug Administration for the treatment of cutaneous T cell lymphoma, and these fusion protein toxins are currently being used to probe the molecular mechanisms by which the catalytic domain of the toxin is delivered from the early endosome to the cytosol of target cells.
- White, A., X. Ding, J.R. Murphy & D. Ringe. 1998. Structure of metal ion-activated diphtheria toxin repressor / tox operator complex. Nature 394:502-506.
- Sun, L., vanderSpek, J., Murphy J.R. 1998. Isolation and characterization of positive dominant mutants of the diphtheria toxin repressor, DtxR. Proc Natl Acad. Sci. USA 95: 14985-14990.
- Twigg, P.D. Wylie, G.P. wang, G., Caspar, D.L.D., Murphy, J.R. Logan, T.M. 1999. Expression and assignment of the 1H, a5N and 13C resonances of the C-terminal domain of the diphtheria toxin repressor. J. Bilmol NMR. 13:197-198.
- Wang, G., Wylie, G.P., Twigg, P.D. Caspar, D.L.D., Murphy, J.R., and Logan, T.M. 1999. Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein. Proc Natl Acad. Sci. USA 96:6119-6124.
- Manabe, Y.C., saviola, B.J., Sun, L., Murphy, J.R. and Bishai, W.R. 1999. Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor. Proc Natl Acad. Sci. USA 96:12844-8.
- Cosenza, L., Rosenbach, A., White, J.V. Murphy, J.R. and Smith, T. 1999. An atomic scale model of interleulin-7 constructed from homologous extension using inverse protein fold predictions and disulfide bond assignments. Protein Science 9:916-26.To see additional publications by investigator click on any article and enter last name and initials in Query box.