X-ray crystallography of proteins. Studies of protein structure and function/enzyme mechanisms and protein-ligand and protein-protein interaction

Our interests are generally in the relationship of protein three-dimensional structure to chemical function. To this end, research is focussed on the modification of the catalytic properties of a number of pharmaceutically or industrially important enzymes. The methods used are a combination of X-ray crystallography, design of transition-state analog inhibitors, and site-directed mutagenesis. The objectives are to learn how to re-engineer these catalysts to perform useful chemical reactions which may not occur efficiently with the naturally occurring enzyme, to dissect the individual steps in a mechanism and characterize them structurally, or to learn how to inhibit an enzyme specifically and selectively.

The proteins being studied currently include enzymes utilizing pyridoxal phosphate as cofactor, a GTP-binding protein, a DNA-binding protein, and several proteases. Different methods are being used to study these systems, including traditional kinetic and structural methods, and low-temperature and time-resolved x-ray structural methods. In addition, a new method for mapping of binding surfaces on proteins is being developed for the design of specific inhibitors.

Pyridoxal is capable of catalyzing several types of transformations. However, any one enzyme utilizing this cofactor does only one of them predominantly. The question therefore arises how the protein controls the chemical outcome of such transformations. We are studying a number of these enzymes structurally in order to begin to answer that question.

The expression of diphtheria toxin in toxigenic Corynebacterium diphtheria is controlled by a transition metal ion activated repressor DtxR. The repressor binds DNA after activation by the metal ion and thereby regulates expression of the toxin. The mechanism of activation is being studied structurally.

Serine proteases are important in cellular development, blood clotting, and a variety of defense mechanisms. Disorders involving these proteases are often linked to the absence or inefficiency of a specific inhibitor to control the activity of the enzyme. The design of such inhibitors requires detailed knowledge of the structure of the enzyme, of the enzyme complexed with inhibitors, and if possible, an understanding of the mechanisms of inhibition

Naffin-Olivos, J.L., Daab, A., White, A., Goldfarb, N., Milne, A.C., Liu, D., Dunn, B.M., Rengarajan, J., Petsko, G.A., Ringe, D.. "Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)." Biochemistry (2017): In Press. (forthcoming) Ringe, Dagmar. "Evidence for Remote Residue Involvement in catalysis: Are Enzyme Active Sites Built in Multiple Layers?." J. Mol. Biol. (2010). (forthcoming) Ringe, Dagmar. "Detection of ligand hot spots on protein surfaces via fragment-based methods: Application toDJ-1 and Glucocerebrocidase.." J. Computer Aided Molecular Design (2009). (forthcoming) Ringe, Dagmar. "The effects of pH and iminosugar pharmacological chaperones on lysosomal gycosidase structure and stability.." Biochemistry (2009). (forthcoming) Ringe, Dagmar. "Novel Inhibitors of the di-Zinc metalloprotease from Vibrio proteolyticus based on a Tris Scaffold." Biochemistry (2007). (forthcoming) Ringe, Dagmar. "Prediction of Interaction Sites from Apo 3D Structures when the Holo Conformation is different." Proteins: Structure, Function and Bioinformatics (2007). (forthcoming) Deshpande, A.R., Pochapsky, T.C., Petsko, G.A., Ringe, D.,. "Dual chemistry catalyzed by human acireductone dioxyge." Protein Eng Des Sel doi: 10.1093/protein/gzw078 (2017): 1-8 [Epub ahead of print]. Wu,R., Sanishvilli, R., Belitsky, B.R., Juncosa, J.I., Le, H.V., Lahrer, H.J.S.,Farley, M., Silverman, R.B., Petsko, G.A.,Ringe, D., Liu, D.,. "PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation." Proc. Natl. Acad. Sci. USA (2017) EE 27. March 2017 (2017). Zahniser, M.P.D., Prasad, S., Kneen, M.M., Petsko, G.A., Ringe, D., McLeish, M.J.,. "Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the class 3 aldehyde Dehydrogenase superfamily." Prot Eng Des Sel doi: 10.1093/protein/gzx015. [Epub ahead of print] (2017): 9:1-8. Bassil, F., Fernagut, P.-O., Bezard, E., Hoang, Q.Q., Ringe, D., Petsko, G.A., Meissner, W.G.. "Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy." Proc. Natl. Acad. Sci. USA 113. doi/10.1073/pnas. 1609291113 (2016): 9593 – 9598. Deshpande et al. "Metal-dependent function of a mammalian acireductone dioxygenase." Biochemistry in press. (2016). Liu, C.F., Brandt, G.S., Hoang, Q.Q., Naumova, N., Lazarevic, V., Hwang, E.S., Dekker, J, Glimcher, L.H., Ringe. D., Petsko, G.A.. "Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites." Proc. Natl. Acad. Sci. USA 113(43). doi/10.1073/pnas.1613914113 (2016): E6572-E6581. Wang, W., Nguyen, L.T.T, Burlak, C., Chegini, F., Guo, F., Chataway, T., Ju, S., Fisher, O., Miller, D.W., Datta, D., Wu, F., Wu, C-X., Landeru, A., Wells, J.A., Cookson, M.R., Boxer, M.B., Thomas, C.J., Wei, P. G., Ringe, D., Petsko. "Caspase-1 causes truncation and aggregation of the Parkinson’s disease-associated protein α-synuclein." Proc. Natl. Acad. Sci. USA 113. doi/10.1073/pnas.1610099113 (2016): 9587– 9592. Berman, D.E., Ringe, D., Petsko, G.A., Small, S.A.. "“The Use of Pharmacological Retromer Chaperones in Alzheimer’s Disease and other Endosomal-related Disorders.”." Neurotherapeutics 12. (2015): 12-18. DOI 10.1007/s13311-014-0321-y. Brodkin, H.R., DeLateur, N.A., Somarowthu, S., Mills, C.L., Novak, W.R., Beuning, P.J., Ringe, D., Ondrechen. M.J.. "“Prediction of Distal Residue Participation in Enzyme Catalysis”." Protein Science in press. (2015). Auclair JR, Johnson JL, Liu Q, Salisbury JP, Rotunno MS, Petsko GA, Ringe D, Brown RH Jr, Bosco DA, Agar JN.. "Post-translational modification by cysteine protects Cu/Zn-superoxide dismutase from oxidative damage.." Biochemistry 52. (36) (2014): 6137-44. Jackson, K.L., Dayton, R.D., Orchard, E.A., Ju, S., Ringe, D., Petsko, G.A., Maquat, L.E., Klein, R.L.. "“ Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.”." Gene Therapy 00. (2014): 1-9 (doi:10.1038/gt.2014.101). Keedy, D.A., van den Bedem, H., Sivak, D.A., Petsko, G.A., Ringe, D., Wilson, M.A., Fraser, J.S.. "Crystal Cryocooling Distorts Conformational Heterogeneity in a Model Michaelis Complex of DHFR." Structure 22. (2014): 899-910. Mecozzi, V., Berman, D.E., Simoes, S., Vetanovetz, C., Awal, M., Patel, V.M. Schneider, R.T., Petsko, G.A., Ringe, D., Small, S.A.,. "“Pharmacological chaperones can stabilize the retromer complex and traffic the amyloid-precursor protein away from intracellular sites of pathogenic processing“." Nat Chem Biol 10. (2014): 443-449. Naffin-Olivos, J.L., Georgieva, M., Goldfarb, N., Madan-Lala, R., Dong, L., Bizzell, E., Valinetz, E., Brandt, G.S., Ringe, D., Dunn, B.M., Petsko, G.A., Rengarajan, J.. "Mycobacterium tuberculosis Hip1 modulates macrophage responses through proteolysis of GroEL2”." PLoS Pathogens 10. (2014): e1004132. Auclair JR, Johnson JL, Liu Q, Salisbury JP, Rotunno MS, Petsko GA, Ringe D, Brown RH Jr, Bosco DA, Agar JN. "Post-translational modification by cysteine protects Cu/Zn-superoxide dismutase from oxidative damage." Biochemistry 52. (36) (2013): 6137-44. Edayathumangalam R1, Wu R, Garcia R, Wang Y, Wang W, Kreinbring CA, Bach A, Liao J, Stone TA, Terwilliger TC, Hoang QQ, Belitsky BR, Petsko GA, Ringe D, Liu D.. "Crystal structure of Bacillus subtilis GabR, an autorepressor and transcriptional activator of gabT." Proc Natl Acad Sci U S A. 110. (44) (2013): 17820-5. Liu, CF, Liu, D, Momb, J, Thomas, PW, Lajoie, A, Petsko, GA, Fast, W, and Ringe D. "A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-y-lactonase from Bacillus thuringiensis." Biochemistry 52. (9) (2013): 1603-10. Sigala PA, Fafarman AT, Schwans JP, Fried SD, Fenn TD, Caaveiro JM, Pybus B, Ringe D, Petsko GA, Boxer SG, Herschlag. "Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site." Proc Natl Acad Sci U S A 110. (28) (2013): E2552-61. Steele JW, Lachenmayer ML, Ju S, Stock A, Liken J, Kim SH, Delgado LM, Alfaro IE, Bernales S, Verdile G, Bharadwaj P, Gupta V, Barr R, Friss A, Dolios G, Wang R, Ringe D, Fraser P, Westaway D, St George-Hyslop PH, Szabo P, Relkin NR, Buxbaum JD, Glabe C. "Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model." Mol Psychiatry 18. (8) (2013): 889-97.