June 1, 2015

High-Resolution Imaging Technique May Advance Drug Design

At a Glance

  • Scientists resolved the detailed architecture of an inhibitor bound to an enzyme in solution.
  • The technical advance could potentially aid drug development in the future.

Imaging with cryo-electron microscopy in near-atomic detail showing the architecture of a metabolic enzyme bound to a drug that blocks its activity.

Many drugs work by fitting into a specific site on a protein, either blocking protein function by acting as an “inhibitor” or by locking it in an “on” position. Determining the detailed 3-D structure of a protein at a very fine level is important in drug development, as it reveals key details about how the drug and protein interact. Understanding these details can allow scientists to design new drugs that either block a protein’s function (if the function is responsible for a disease), or enhance its function (if lack of activity is causing a problem).

A research team led by Dr. Sriram Subramaniam of NIH’s National Cancer Institute (NCI) has been working to determine the structure of proteins at high resolution using an imaging technique called cryo-electron microscopy (cryo-EM). Until recently, the highest resolution protein structures were determined by X-ray crystallography, a technique that requires a protein to first be crystallized into a fixed 3-D shape. Cryo-EM doesn’t require crystallization, but hasn’t previously achieved resolutions that can visualize individual atoms within a structure.

In their new study, the team used cryo-EM to determine the structure of a small bacterial protein called beta-galactosidase, which functions as an enzyme in the cell. A drug called phenylethyl-beta-D-thiogalactopyranoside (PETG) fits into a pocket in the enzyme to turn it off.

The researchers rapidly froze a mixture of the enzyme and drug at an extremely low temperature (about -196 °C to -210 °C, or -320 °F to -346 °F). The technique, called plunge freezing, quickly stabilizes the water around proteins without allowing damaging ice crystals to form. This approach keeps proteins in their natural form and protects them during imaging. Using an electron microscope, the team then examined the structure of the enzyme alone and with the drug. Results were published online on May 7, 2015, in Science.

The team combined more than 40,000 molecular images to build a map of the structure of beta-galactosidase bound to PETG at a resolution of about 2.2 angstroms (about a billionth of a meter in size). They not only determined how PETG fits into the protein, but also mapped individual water molecules and ions within the structure. This type of detail is what’s needed to understand drug-protein interactions and aid in drug development.

“The fact that cryo-EM technology allows us to image a relatively small protein at high resolution in a near-native environment, and knowing that the structure hasn’t been changed by crystallization, that’s a game-changer,” Subramaniam says.

The group believes that cryo-EM may become a key tool to reveal the structure of proteins and thus to assist in drug design and development efforts.

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Reference: 2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Bartesaghi A, Merk A, Banerjee S, Matthies D, Wu X, Milne JL, Subramaniam S. Science. 2015 May 7. pii: aab1576. [Epub ahead of print]. PMID: 25953817.

Funding: NIH’s National Cancer Institute (NCI) and Office of the Director (OD).