Diagram of full length KcsA in a lipid bilayer. Note how much is missing from the crystal structure.

Mobile regions of membrane proteins

Membrane proteins are essential components of all organisms and are critical drug targets. Although we’ve made major gains in determining the structure of this type of protein, we have few tools that can resolve the portions of membrane proteins that extend past the membrane, that is loop and termini regions. These regions are the portions that allow the protein to sense changes in its environment and are generally the sites where drugs dock. Much of my research is focused on developing methods to resolve the structures of these regions.

All organisms are made of cells. A complex mixture of lipids, proteins and many other components make up the membrane, the shell, that encloses each cell. Potassium channels are a category of protein that selectively allow potassium ions to cross from one side of the membrane to the other.

KcsA is a channel that allows potassium ions to flow into the cell of a bacterium when the interior of that cell becomes acid, that is when the pH is decreased. And so we call KcsA an inward rectifying pH-gated potassium channel. This particular channel comes from the soil bacteria Streptomyces lividans. We don’t know why a bacteria needs such a channel. A good hypothesis is that certain metabolic processes can cause a cell’s pH to drop. This change affects the operation of the cell, interfering with many of the normal operations of a cell, inactivating important proteins, for example. So, to cope with such a scenario, to cope with the stress of a decreased pH, KcsA opens up and let’s potassium flood into the cell. The potassium itself does nothing to help with the acidic environment directly, but the additional potassium ions alter the charge of the interior of the cell. The change in voltage in the bacterium activates other cell functions, triggering voltage-gated channels and other proteins. All of this would contribute to restoring the bacteria’s pH back to a normal level where the cell operates most efficiently.

The organism Streptomyces lividans isn’t of much medical value, being a nonpathogenic bacteria. So why study KcsA? Part of it is luck, it happened to be one of the first ion channels to be identified, then its sequence, then finally in 1999, its atomic-level structure, work that would win Roderick MacKinnon a Nobel Prize for the first x-ray crystal structures of ion channels, of KcsA. So in part, we study it because, well, we’ve studied it. We stand on the shoulders of giants and there’s so many deeper questions that we can ask with all of the accumulated information.

KcaA x-ray crystal structure 1BL4. Science 280, 69–77 (1998).

Since it is a protein, KcsA is comprised of a linear sequence of chemical groups called amino acids, of which there are twenty types. We can think than of a protein as a chain where each link can be any of the twenty types of link. KcsA is comprised of 160 of these links, and to be a KcsA it must, by definition be a specific chain of 160 amino acids in a particular order. KcsA channels are then twist themselves into interesting characteristic shapes, and four of these chains all ranged together make one single channel. The reason that determining the structure, the way the chain characteristically shapes itself, can reveal a great deal about the function of the protein. Structural data can also be useful in order to develop drugs that might act on a particular channel, temporary interfering with its normal function. In fact, nearly 90% of all drugs act on protein targets that are bound to the membrane, just like KcsA. So, determining the structure of KcsA was interesting in its own right. But it also heralded a new era, one in which scientists could collect and use the structural data on an entire class of vitally important proteins.

Since the original structure was published, dozens of unique KcsA (representing a host of unique conditions and techniques to acquire the structure) and hundreds of other membrane proteins. Yet despite these gains, we still have few reliable tools that can map the regions of membrane proteins that extend past the membrane itself. These loop regions and the termini are often the portions of membrane proteins responsible for responding to the environment. For KcsA, the loops and the termini sense the pH and the potassium concentration in the cell. When the pH inside the cell drops, that is when the cell becomes dangerously acid, the KcsA activates and allows potassium to flood into the cell. It’s believed that this initiates a cascade of events within the cell that lets the bacteria restore a healthy pH.

Diagram of full length KcsA in a lipid bilayer. Note how much is missing from the crystal structure.

 

It turns out the the loop and tail regions tend to interfere with techniques that produce good crystals. So, researchers tend to cut off those bits in order to solve their structures. Much of my work studies membrane proteins by solid-state nuclear magnetic resonance. That technique is best suited for studying rigid portions of proteins, the bits that don’t tend to move much, such as the domains trapped in the membrane. The tails and the loops are constantly moving, flexing and shifting, which means that those regions tend to be silent in solid-state studies. So despite the incredible power of crystallography and solid-state NMR, there’s still plenty of work to be done to look at the loopy bits.