Dorothee Kern, Ph.D. Professor of Biochemistry Investigator, Howard Hughes Medical Institute Structure, Dynamics and Function of Enzymes Ph.D. Martin-Luther-University Halle, Germany
	contact information (781) 736-2354

Motions are the basis for the function of biological macromolecules. Whereas for many enzymes the structures have been solved, very little is known about dynamic processes, which trigger catalysis. Our main goal is to characterize these motions in enzymes to provide a better understanding of the atomic events in enzyme catalysis as a function of time. This knowledge will be used for the design of highly efficient inhibitors. We use NMR as a primary tool because it is an excellent method to measure dynamic processes ranging from picoseconds to days. Our current work is focused on three topics:

1	Correlation between protein dynamics, enzyme kinetics and function;
2	Structural basis of activation of response regulators in signal transduction triggered by phosphorylation;
3	Mechanism of Thiamin diphosphate (ThDP)-mediated enzyme reactions.

1. To analyze the kinetics of reversible enzyme reactions we developed a new technique using NMR line shape analyses of the substrate in the presence of catalytic amounts of enzyme. To understand how the enzymes can carry out catalysis we measure backbone and side-chain dynamics in "working" enzymes. We recently succeeded in measuring the dynamic "hotspots" during catalysis using human cyclophilin A as the first model system Figure 3 (Eisenmesser et al,. Science 2002, 295, 1520-3). The rates of conformational dynamics of the enzyme (microseconds) strongly correlate with the microscopic rates of substrate turnover. The ultimate goal using this novel approach will be to "see" macromolecules reacting in a "real-time movie". For this purpose the experimental data are used in combination with computational methods. We are currently working with enzymes that are highly interesting for medicine because they are essential for HIV infectivity or cell cycle control.
   
   
2. Two-component regulatory systems are the dominant molecular switches in bacterial signaling and have recently been found in eukaryotes. The two components are a histidine kinase and a response regulator, which are activated via phosphorylation. The very short half-lives of the aspartyl-phosphate linkages in active response regulators made structural studies extremely difficult. Recently we succeeded to solve the first structure of a receiver domain in its active state by applying unconventional NMR spectroscopy (Kern et al, Nature (1999) 402, 894-898): To determine a structure of the transiently phosphorylated protein (lifetime of 2 minutes) at a concentration as low as 0.3 mM, NMR spectra were taken on a working enzyme and multiple three dimensional spectra were added. Phosphorylation induces a large conformational change (Figure 1). Second, we are investigating the kinetics of the activation using NMR relaxation experiments. Surprisingly we found that both the inactive and active conformation is already populated before phosphorylation. Phosphorylation shifts a pre-existing equilibrium. We propose that shifts of pre-existing equilibria may be a fundamental paradigm of ligand binding and signaling. Figure 2 shows a movie presentation of the unphosphorylated form of NtrC (Volkman et al., Science 2001, 291, 2429-33). Our next goal is to unravel how activation is transmitted to the downstream target, the transcriptional activation domain. For this purpose the protein splicing enzymes inteins are used to segmental label domains.
   
   
3. We are using NMR spectroscopy to answer a question of biochemistry, which has been controversial for decades: How is thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, activated in enzymes? Although ThDP catalyzes a wide range of functions in different enzymes, the first step in catalysis is common in all ThDP dependent enzymes. We were able to unravel the mechanism of activation by combining quenched flow experiments with NMR, synthesis of cofactor analogues and mutagenesis. This is an example of how NMR can be applied even on large proteins (240 kDa) to study enzyme mechanisms. We are trying to investigate this new proton shuttle reaction in more detail.




Figure 1: Molecular switch upon phosphorylation of Asp 54 in the response regulator NtrCr. Ribbon structures of the phosphorylated, active conformation (yellow/orange) and the unphosphorylated, inactive form (cyan/blue) were superimposed using residues indicated in darker colors (orange and blue). The regions of greatest difference are highlighted (yellow and cyan, the "switch-area"). Upon phosphorylation of Asp 54, b-Strands 4 and 5 and a-helices 3 and 4 tilt to the left - away from the active site. In addition, a register shift by about two amino acid residues from the N- to the C-terminus and a rotation by about 100o about the helical axis are induced in helix 4 upon phosphorylation. The rotation results in a change in orientation of the hydrophobic side-chains in helix 4 from the inside to the outside.




Figure 2: Mechanism of activation investigated by backbone dynamics experiments. Before phosphorylation, both inactive and active states are existing, and phosphorylation shifts this pre-existing equilibrium towards more than 95% active conformers. The movie illustrates the constant interconversion between the states (microsecond lifetime of the two states). Volkman et al., Science 2001, 291, 2429-33

Movie




Figure 3: Enzyme dynamics during catalysis of human cyclophilin A. (A) Structure of the cis conformation of the substrate Suc-Ala-Phe-Pro-Phe-4-NA (green) bound to CypA. CypA residues with chemical exchange in both the presence and absence of substrate are color coded in blue. Residues in red exhibit chemical exchange only during turnover. Residues shown in magenta exhibit chemical exchange in the absence of substrate, but increase in its presence. (B) Suggested trajectory of the enzymatic pathway based on the dynamics results. CypA catalyzes prolyl isomerization by rotating the part C-terminal to the prolyl peptide bond by 180¡ to produce the trans conformation of the substrate. In this model, the observed exchange dynamics for residues in strand 5 can be explained. Eisenmesser et al,. Science 2002, 295, 1520-3

Selected Publications:

Henzler-Wildman KA. and Kern D. “Dynamic personalities of proteins.” Nature. (2007) 450, 964-972

Henzler-Wildman KA, Thai V, Lei M, Ott M, Wolf-Watz M, Fenn T, Pozharski E, Wilson MA, Petsko GA, Karplus M, Hübner CG, Kern D.” Intrinsic motions along an enzymatic reaction trajectory.” Nature (2007) 450, 838-844.

Henzler-Wildman KA, Lei M, Thai V, Kerns SJ, Karplus M, Kern D. “A hierarchy of timescales in protein dynamics is linked to enzyme catalysis.” Nature. (2007), 450: 913-916.

Eisenmesser, E.Z., Millet, O., Labeikovsky, W., Korzhnev, D.M., Wolf-Watz, M., Bosco, D.A., Skalicky, J.J., Kay, L.E. and Kern, D. "Intrinsic dynamics of an enzyme underlies catalysis." Nature. (2005), 438, 117-121. [abstract]

Kern, D., Eisenmesser, E.Z. and Wolf-Watz, M. "Enzyme dynamics during catalysis measured by NMR spectroscopy." Methods in Enzym. (2005), 394, 507-524. [abstract]

Wolf-Watz, M., Thai, V., Henzler-Wildman, K., Hadjipavlou, G., Eisenmesser, E.Z. and Kern, D.) "Linkage between dynamics and catalysis in a thermophilic mesophilic enzyme pair." Nat. Struct. Mol. Biol. (2004) [PubMed Abstract]

Kern D, Zuiderweg ER. "The role of dynamics in allosteric regulation." Curr Opin Struct Biol. (2003) 13(6):748-57. [PubMed Abstract]

Eisenmesser E.Z., Bosco D.A., Akke M., and Kern D. "Enzyme Dynamics During Catalysis." Science. (2002) 295:1520-3 [Full Article] [PubMed Abstract] Prof. Joseph J. Falke has written a perspective on this article.

Bosco, D.A., Eisenmesser, E.Z., Pochapsky, S., Sundquist, W.I. and Kern, D. "Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A." Proc. Natl. Acad. Sci. U.S.A. (2002). 99(8), 5247-5252 [PubMed Abstract].

Volkman BF, Lipson D, Wemmer DE, Kern D. "Two-State Allosteric Behavior in a Single-Domain Signaling Protein." Science. (2001) 291, 2429-2433. [PubMed Abstract] [Full Article] [Movie]

Kern, D., Volkman, B.F., Luginbühl, P., Nohaile, M.J., Kustu, S., and D.E. Wemmer. "Structure of a transiently phosphorylated "switch" in bacterial signal transduction", Nature, (1999) 402, 894-898. [PubMed Abstract]

Kern, D., Kern, G., Neef, H., Tittmann, K., Killenberg-Jabs, M., Wikner, C., Schneider, G., & Hübner, G. "How thiamin diphosphate is activated in enzymes", Science, (1997) 275, 67-70.

View Complete Publication List on PubMed: Dorothee Kern

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