Cryo-EM and the Structure of TRPV1
Updated: Oct 4, 2019
Recent Advances in Cryo-Electron Microscopy
Protein structures are important in understanding the molecular basis for their mechanisms of action, without structures the binding site of a signalling molecule for example might be unknown. A structure therefore also allows you to design novel molecules that could bind to your protein of interest, potentially changing its activity. This process is called “Structure Based Drug Design” and is responsible for thousands of the drugs that we use today. The resolution of protein structures is a measure of the quality of the model, this is represented by a size. For example, the structure that will follow in this article was determined to a resolution of 3.4 Å (One Ångstrom is equal to 10−10 m, or in other words, one ten-billionth of a metre!).
X-ray crystallography is seen by many scientists as the gold standard of structural biology. It is a technique that can uncover high-resolution (generally under 5 Å) structures of a protein by directing a beam of X-rays through a crystal of homogenous protein and recording the resulting diffraction pattern. It is an excellent technique however it can be extremely difficult to obtain crystals from your protein sample, this is especially true with membrane proteins which require detergents to cover hydrophobic regions of the protein or proteins with dynamic conformations. In some cases it is simply not possible to crystallise a protein and another structural technique must be used.
Cryo-electron microscopy (cryo-EM) is a technique also commonly used in structural biology. Rather than having to produce crystals for analysis, cryo-EM can directly scan over a surface that is covered with thousands of individual protein molecules. It is able to overcome many of the challenges of other structural techniques such X-ray crystallography but until recently it has proved difficult to produce high-resolution structures. Small proteins could only be reconstructed to much lower-resolutions that X-ray crystallography resulting literally in blob-like structures (My supervisor actually refers to cryo-EM as “blobology”). This limitation was due in part to how electron microscopes worked. After hitting the target sample with a stream of electrons, the reflected electrons would then strike a phosphor-coated screen, which in turn would emit a photon that could be detected by a CMOS camera. A newly developed technology is able to detect electrons directly, significantly improving the quality of data that is recorded.
Liao et al. from the University of California, made use of this new technology along with a few extra tricks discussed later to uncover the first ever atomic-level structure of TRPV1.
Transient receptor potential (TRP) Channels
TRP channels such as the mammalian receptor TRPV1 are proteins that create small channels in the cell membrane, allowing cations to pass through when activated by specific stimuli. Generally ion channels are involved in signalling pathways and the influx of ions leads to the activation of downstream signalling partners.
TRPV1 channels are activated by capsaicin (the molecule responsible for the heat in chillis). They are also part of a sub-family of TRP channels that allow specific cell types to detect changes in local ambient temperate. Noxious heat, modulated by inflammatory agents, can activate TRPV1, leading to pain sensitivity. TRP channels therefore are considered as important drug targets due to their roles in pain physiology and a three-dimensional structure would prove extremely valuable.
The Structure of TRPV1
When expressing a protein for structural studies it is advantageous to select for a subtype that has a high stability if possible. In this case, Liao et al. identified a mutant rat TRPV1 channel that maintained most wild-type properties whilst having increase stability. After cloning, the protein was expressed in HEK293 cells and purified ready for cryo-EM. New image-processing algorithms were used to correct for motion-induced blurring of each single-particle cryo-EM image. The resulting map of TRPV1 (Figure 1b) was at a resolution high enough for de novo atomic model building. This is the process in which the polypeptide chain is fitted inside the cryo-EM map to give a protein structure (Figure 2).
In addition to the closed structure (Figure 2), two open structures were determined. In one open structure, capsaicin is bound and in the other are two potent activators, resiniferatoxin and double-knot toxin. These new structures will allow scientists to design new TRPV1-specific drugs and investigate different methods of TRPV1 regulation.
The approach used to generate these structures is extremely powerful and useful in cases where X-ray crystallography is not an option. Liao et al. have paved the way for many new and exciting structures to come.
Structure of the TRPV1 ion channel determined by electron cryo-microscopy M. Liao and E. Cao and D. Julius and Y. Cheng Nature 504 107-12 (2013) Single particle electron cryo-microscopy of a mammalian ion channel M. Liao and E. Cao and D. Julius and Y. Cheng Curr Opin Struct Biol 27C 1-7 (2014) Structure of TRPV1 channel revealed by electron cryomicroscopy V. Y. Moiseenkova-Bell and L. A. Stanciu and I. I. Serysheva and B. J. Tobe and T. G. Wensel Proc Natl Acad Sci U S A 105 7451-5 (2008)