79 research outputs found
Evolution of protein dynamics over 3.5 billion years at the heart of enzyme catalysis and regulation
As a direct manifestation of molecular kinetic energy, temperature is a fundamental evolutionary driver for chemical reactions. However, it is currently not understood how the natural evolution of catalytic efficiency responds to dramatic changes in environmental temperatures. Using Ancestral Sequence Reconstruction (ASR) we resurrect and biophysically characterize the oldest common ancestral kinase and enzymes along the evolutionary path to modern kinases. Strikingly, enzymes coped with an inherent drop in catalytic speed caused as the earth cooled down over 3.5 billion years by accelerating protein dynamics and adapting thermostability by unexpected mechanisms, as characterized by NMR. Tracing the evolution of enzyme activity and stability from the hot-start towards modern hyperthermophilic, mesophilic and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level (1). In the second part of my talk I will describe our experimental exploration of the evolution of two allosteric regulation mechanisms widely found in the modern protein kinase superfamily, phosphorylation of the activation loop and binding of a regulatory partner protein. The results reveal the origins of allosteric activation including surprising mechanistic features. Moreover, ASR enabled identification of the underlying allosteric network in modern kinases that spans from the N-terminal to the C-terminal lobes. We are currently exploiting this knowledge for the development of allosteric inhibitors and activators. This latter approach delivered novel kinase inhibitors and activators with extreme specificity and high affinity thereby opening the road to new cancer treatment.
(1) V. Nguyen, C. Wilson, M. Hoemberger, J. Stiller, R. Agafonov, J. English, S. Kutter, D. Theobald and D. Kern “Evolutionary Drivers of Thermoadaptation in Enzyme Catalysis” Science 2017, 35
Conformational pathways of Adenylate Kinase characterized by computations, pressure and experiments
Enzyme dynamics during catalysis
Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory
Altered Cytokine Expression and Barrier Properties after In Vitro Infection of Porcine Epithelial Cells with Enterotoxigenic Escherichia coli and Probiotic Enterococcus faecium
The aim of the present study was to elucidate the effects of the probiotic
feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal
epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic
Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both,
and the effects on barrier function and structure and intra- and intercellular
signaling were determined. Coincubation with E. faecium abolished the ETEC-
induced decrease in transepithelial resistance (Rt). No differences were seen
in the expression levels of the intercellular connecting tight junction
proteins examined. However, for the first time, a reorganization of the
monolayer was observed in ETEC-infected cells but not in coincubated cells.
ETEC induced an increase in cytotoxicity that was prevented by coincubation,
whereas apoptosis rates were not affected by bacterial treatment. ETEC
increased the mRNA expression and release of proinflammatory cytokines TNF-α,
IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA
expression and IL-6 protein. Likewise, cAMP concentrations elevated by ETEC
were reduced in coincubated cells. These findings indicate a protective effect
of the probiotic E. faecium on inflammatory responses during infection with
ETEC
Dynamics of human protein kinase Aurora A linked to drug selectivity
Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome
Altered Cytokine Expression and Barrier Properties after In Vitro Infection of Porcine Epithelial Cells with Enterotoxigenic Escherichia coli
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