Home › Research › Snapshots of Metalloenzymes in Action
Our Scientists
Snapshots of Metalloenzymes in Action
Research Summary
The Drennan laboratory seeks to understand how nature harnesses and redirects the reactivity of enzyme metallocenters to perform challenging reactions. By combining x-ray crystallography with other biophysical methods, their goal is to "visualize" molecular processes by obtaining snapshots of enzymes in action.
Why Snapshots?
Suppose you want to understand the process by which a mechanic changes the battery in your car. One photo of a mechanic standing by the car won't do the trick, but a series of photos of the mechanic changing the battery would be informative. The same is true when you want to understand a cellular process; one image of a key enzyme tells you only so much. Thus, our laboratory uses multiple biophysical methods to aid in the "visualization" of complex enzyme processes.
Which Enzymes Are the Subjects of Our Snapshots?
As chemists, we want to understand how nature performs the most challenging chemical reactions. Often, these reactions require the use of organic cofactors or metal ions, which enhance enzyme reactivity. Thus, metallo- and cofactor-dependent enzymes are our primary targets. For example, the reaction of class I ribonucleotide reductase (RNR) utilizes a di-iron cofactor to generate protein-based radical species, essential for the reduction of ribonucleotides to deoxyribonucleotides. Although enzymes like RNR are important and fascinating to study using structural methods, their structural characterization is often non-trivial due to issues such as conformational flexibility, protein heterogeneity, structural complexity, and oxygen sensitivity. Our laboratory has specialized in tackling and solving these challenging structural biology problems.
Figure 1: Two molecules of a corrinoid iron-sulfur protein (CFeSP) form a complex with a dimeric methyltransferase (MeTr2). MeTr2 binds CH3-H4folate and transfers its methyl group to the B12cofactor of CFeSP. In this structural snapshot, B12 is too far from the folate-binding site for methyl transfer (~18 Å, suggesting that a large conformational change must occur. Molecules of CFeSP are in green/orange/cyan and purple/orange/blue; MeTr2 is in pink/red; B12 is shown as magenta sticks, Fe as orange spheres, and S as yellow spheres.
Figure 2: Mechanical disruption of nucleosomes by stretching DNA. A: A single DNA molecule The prototypic RNR from E. coli exists as free subunits (left), a compact “active” α2β2 state (middle), and a doughnut-like dATP-inhibited α4β4 form (right). Ab initio small-angle x-ray scattering reconstructions are fit with crystal structures of α2 (blue/green) and β2 (red/orange). X-ray crystallography and electron microscopy also support the α4β4 structure.
Ando, N., Brignole, E.J., Zimanyi, C.M., Funk, M.A., Yokoyama. K., Asturias, F.J., Stubbe, J., and Drennan, C.L. 2011. Proceedings of the National Academy of Sciences USA 108:21046–21051.
In addition to our focus on radical-based enzymology, we also study metallo- and cofactor-containing enzymes involved in carbon dioxide sequestration and methylation chemistry. In this research, we seek to obtain snapshots to understand how one-carbon units such as CO2 or CH3 are transferred from enzyme to enzyme. We recently presented the long-awaited x-ray structure of all protein components required for the methyl transfer from folic acid (vitamin B9) to vitamin B12 (Figure 1), demonstrating that the methyl transfer between these B vitamins requires a large conformational change promoted by folate binding (Movie 1 and 2). Our laboratory is also broadly interested in how metallocofactors are assembled on their target enzyme and in the regulation of metal uptake by cells.
How Do We Take Snapshots?
Although our main technique is x-ray crystallography, we have recently expanded our toolbox and now combine crystallographic results with data from a variety of other methods, including small-angle x-ray scattering (SAXS), electron microscopy (EM), analytical ultracentrifugation (AUC), computational biophysics, absorbance spectroscopy, and nuclear magnetic resonance (NMR). The value of this combined approach is exemplified in our recent work on the prototypic class I RNR from Escherichia coli. Despite the importance of RNRs as antitumor, antiviral, and antibiotic drug targets, structural information was restricted for decades to isolated depictions of α2 and β2 subunits. To determine the first structure of a RNR holocomplex, we combined x-ray crystallography with SAXS, EM, and AUC. We found that under physiological conditions, this prototypic RNR exists as a mixture of oligomeric species, an active α2β2state, and an inhibited α4β4 state, whose distributions are modulated by ATP/dATP ratios (Figure 2). The unprecedented α4β4 doughnut-like inhibited state is flexible enough to break apart to form a compact α2β2 active structure when dATP levels diminish (Movie 3). Because of the structural heterogeneity of this enzyme, any single structural technique would not have been able to tackle this problem successfully.
Using this technique toolbox, we strive to unmask the secrets of metalloenzyme reactivity: from unveiling the three-dimensional structures of newly discovered metallocenters to "watching" these enzymes juggle and communicate as they carry out their chemical reactions.
Grants from the National Institutes of Health provided partial support for these projects.
