For the first time, researchers have used X-ray crystallography and NMR to directly visualize an enzyme in its low and  higher-energy state and demonstrated the crucial role of interconversion between these states for catalysis. The study offers up new molecular sites as potential drug targets.

Understanding how enzymes work as catalysts depends on understanding their structures and how these change in response to the presence of their substrate and how those changes in turn cause chemical changes in the substrate itself. Crystal structures have provided detailed information over many years regarding the nature of enzymes and enzymes within which a substrate both natural and non-natural are bound. However, some crucial enzyme energy states have remained hidden from crystallographers and it is such high-energy states that may provide the most important clue as to how an enzyme functions, how it might be controlled pharmaceutically.

Now, Michael Clarkson and Dorothee Kern of the Department of Biochemistry,at the Howard Hughes Medical Institute, at Brandeis University, in Waltham, Massachusetts, working with James Fraser, Sheena Degnan, Renske Erion, and Tom Alber of the Department of Molecular and Cell Biology, at the University of California, Berkeley, report how to see such rare states.

They have, for the first time, used ambient-temperature X-ray crystallography and nuclear magnetic resonance (NMR) techniques to directly visualize protein structures essential for catalysis in the rare high-energy state. The study also reveals the motions of these rare states and shows how they contribute directly to the enzyme's catalytic prowess.

The research builds on pioneering NMR studies at Brandeis by biophysicist Dorothee Kern and her team that linked protein function to an enzyme's rare high-energy state, in the absence of catalysis. That work put to rest the notion that proteins are inactive except when being active.

Now, Kern, Alber and colleagues have pushed the work further by analysing previously discarded electron density data originally considered nothing more than noise in the analysis of human cyclophilin A (CYPA), an enzyme hijacked by HIV during replication. They used a novel algorithm, Ringer, to systematically sample the electron density around each dihedral angle to discover additional unmodelled side-chain conformers.

The key to the success of the study was some clever protein design, through controlled mutation, together with dynamic NMR spectroscopy that provided direct experimental evidence that the hidden structures in the high-energy state are in fact essential for catalysis. The researchers revealed what happens when proteins flip from the rare state to a major state in a process called interconversion. If this flip is rapid, then the enzyme does its job quickly, but if the flip is slow, as in the modified enzyme, then the enzyme operates slowly.

"People always focused on the chemistry - accelerating the reaction through catalysing the chemical step of the substrates," explains Kern. "What we've shown is that protein dynamics is as important as the chemical step." She adds that, "Basically, all the steps need to be choreographed just right, like steps for a beautiful dancer. An enzyme can only function well with the perfect choreography of all the components. We now can show directly that the higher energy states are always there and that these hidden, rare states are absolutely essential for protein function."

"Such knowledge may enable progress not only in understanding and manipulating the mechanisms of numerous macromolecular systems, but also in defining the manifold of conformations accessible to inhibitors and therapeutics," the team concludes.
 

Related links:

• Nature, 2009, 462, 669-673
• Dorethee Kern 

Article by David Bradley