X-ray crystallographic analysis of macromolecular assemblies
1. Transcription regulation in a chromatin context The complete blueprint for each cell is stored in the form of DNA, and the fate of an individual cell depends exclusively on the timed and coordinated readout of the correct DNA regions. The five to six billion DNA base pair steps in the human genome, for example, would stretch over a length of almost 2 meters, but in fact are compacted in the cell nucleus to nearly one millionth of this dimension in the material called chromatin. The extreme compaction of DNA that is necessary to fit it into the confines of the nucleus (in the form of chromatin) has profound implications for our understanding of transcription regulation. Chromatin is built from nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. About 25 million of them are required per nucleus to accomplish this extraordinary compression of DNA. Long arrays of nucleosomes are further coiled up and condensed in a number of hierarchical levels, such as the attachment of large (20-50 kilobase pair) loops to the nuclear scaffold of chromosomes. Chromatin is all the more remarkable because it must enable access to specific regions of the DNA molecule so that only a relatively small number of genes are read and used, the rest remaining repressed. At other times, access over the entire genome must be permitted so that all of the DNA can be replicated. These processes take place while the histones remain associated with the DNA, preventing the chromatin from becoming significantly unraveled.
Our research is focused on the role of chromatin structure in the regulation of transcription. In particular, we are engaged in investigating the structural basis for three different aspects of this complex problem:
How do transcription factors recognize and bind their DNA target sequence in a chromatin context?
What exactly is the 'remodeled' state of chromatin that appears to be necessary to allow transcription of many packaged genes?
How is the formation of chromatin loops on the nuclear scaffold involved in the regulation of transcription?
We are approaching these questions by determining the three-dimensional structures of the macromolecular complexes in question. X-ray crystallography is the central technique in our lab. Our new X-ray facilities are equipped with a state of the art x-ray generator with multilayer optics and an R-Axis-IV imaging plate. The heart of our computer facilities is a Dec 600au personal workstation, which is suitable for high quality graphics applications as well as for data reduction and refinement. In addition to crystallographic methods, we are also employing fluorescence spectroscopy and other biophysical assays to gain insight into the dynamic behavior of the systems under investigation.
2. The induced-fit mechanism in the antibiotic target MurA and its relationship to the herbicide target EPSP synthase MurA (UDP-N-acetylglucosamine enolpyruvyltransferase, EC 184.108.40.206) initiates the biosynthesis of the heteropolymer murein, the major structural element of the bacterial cell wall. It catalyzes the transfer of the intact enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3-hydroxyl of UDP-N-acetylglucosamine (UDP-GlcNAc). MurA is of potential pharmaceutical interest because it is inhibited by the broad spectrum antibiotic fosfomycin. The only other enzyme known to catalyze a similar reaction is 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 220.127.116.11). EPSP synthase catalyzes the sixth step in the biosynthesis of the aromatic amino acids (the shikimate pathway) in bacteria, plants and fungi and has been extensively studied because it is the target of glyphosate, the active ingredient of the broad-spectrum herbicide Roundup' . Recently, the mechanistic relationship between MurA and EPSP synthase was given a structural basis through the determination of the crystal structure of MurA. Like EPSP synthase, MurA is a two-domain protein with an unusual fold (inside-out a/b barrel) made up by the six-fold repetition of one folding unit. The crystal structures of MurA in ligand-free  and UDPGLcNAc/fosfomycin complexed form  revealed that the reaction apparently follows an induced-fit mechanism in which the two-domain structure undergoes large conformational changes [Fig. 1]. The catalytically essential residue Cys115 is located in a flexible loop, which, in the course of the enzymatic reaction, moves about 20 _ towards the inter-domain section. The large conformational changes upon binding of substrates and/or the naturally occuring inhibitor fosfomycin are predominantly mediated by UDP-GlcNAc . Currently we characterize the dynamics of a number of single-site mutants of MurA by various techniques. In order to understand the structural basis of the induced-fit mechanism, high resolution X-ray crystallographic analysis of mutant proteins with altered dynamic properties is underway. This approach may serve as a starting point to the development of novel antibiotics. In addition, we attempt the crystal structure determination of glyphosate bound to EPSP synthase. To date, the binding-site of glyphosate in EPSPS is unknown and an induced-fit mechanism similar to that of MurA is under question.
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