The structure of a protein has apparently been discovered that seems to pinch off tiny pouches from cells’ outer membranes. The scientists at The Scripps Research Institute and the National Institutes of Health (NIH) claim to have discovered this. It is known that the cells use these pouches, or vesicles, to carry nutrients and other essential substances. Even many medicines seem to take a ride inside them.
This research may aid in answering vital queries of how vesicles form, by highlighting the structure of the protein, dynamin. The experiment bestowed knowledge about the process critical to cell survival. Efficient ways to deliver drugs could also be obtained. It was assumed that the cell membrane behaved as a hurdle around the cell, thus, keeping out all the possible harmful materials. Yet the cells too may require some substances to enter.
However, to pass the membrane, nutrients or hormones in the bloodstream the specific receptors begin binding on cell membranes. A pit is then known to be formed by the membrane around these bound molecules. They further, squeeze into a pouch, or vesicle, that detaches from the rest of the cell membrane and carry their essential cargo into the cell. It seems that the nerve cells utilize this same vesicle-making mechanism, called endocytosis. This helps to maintain signaling from one cell to another.
Sandra Schmid, chair of the Scripps Department of Cell Biology and senior author of the Nature article along with Fred Dyda at NIH announced, “Endocytosis is how cells communicate. It’s critical for many biological functions from controlling blood pressure to getting rid of glucose.”
Though the importance of endocytosis was probably known, the functions of the cells to perform this process remained unknown. The scientists were only aware that this process was initiated by at least one molecule, dynamin. It has been revealed that the dynamin belongs to a large family of enzymes called GTPases. These enzymes release energy when they bind the chemical GTP and convert it into a simpler form GDP. Also, during this conversion itself a GTPase change its shape. This enables it to perform a particular function such as making vesicles.
In the past, there was a contradictory belief among the scientists, that dynamin molecules assembled long spirals on cell membranes. They also held the view that in the presence of GTP these spirals tightened, lopping off a vesicle. However, a research conducted by Schmid’s group claimed to prove that dynamin proteins only form a short collar around the cell membrane. They apparently watched the vesicle transformation through a microscope. They also, claimed that a dynamin can act alone, without the help of any other proteins. Schmid stated that the dynamin is the master regulator of endocytosis and is involved at every stage of vesicle formation.
That research nevertheless failed to reveal as to how the dynamin collars pinch off membrane vesicles. So, Schmid and others began conducting research on dynamin’s GTPase activity for clues of how it controlled the process. It was assumed that a means to known how a protein functions was by determining the structure. To analyze the structure the scientists used a technique called X-ray crystallography. In this technique crystals of the protein of interest were made and then bombarded. This is done with the X-rays to know the position of the atoms.
Apparently, this technique was not effective for dynamin as it was a large molecule. It consisted of almost 1,000 amino acids, making it difficult to crystallize. However, about three years ago, Joshua Chappie, then a graduate student in Schmid’s laboratory, engineered a shorter version of dynamin that retained the same GTPase activity as the complete protein.
Chappie could now quickly obtain crystals and examine them by X-ray crystallography. Disappointingly, the double vision of the X-ray signals made the data unable to interpret. However, Chappie discovered that the minimal dynamin formed dimers during its normal cycle of GTP hydrolysis. It was probably known to the researchers that dynamin was found in cells as a group of four molecules, or a tetramer. The dynamin tetramers were claimed to be held together by long stalk regions with the GTPase domains protruding from their tops. But the minimal dynamin lacked the stalk regions and existed as monomer. The stalk regions lacked the minimal dynamin and existed as a monomer. Functional dimers were probably formed of the GTPase domains of neighboring tetramers by assembling the dynamin in short collars. These functional dimers were essential for not only GTPase activity but also membrane pinching.
The investigators then after through analyses, concluded that, when the GTPase domains from different tetramers paired up, probably the structures of the tetramers shifted. This made them less stable. Another change in the shape was caused in the tetramers during the conversion of GTP to GDP. The outcome was that, the dimers dissociated and the entire collar structure came apart. There was repeated cycle of collar assembly, GTP binding, GTPase domain dimerization, conversion of GTP to GDP, and disassembly of the dynamin collar when vesicle was formed. It is these cycles that in the course of time twisted and pinched off the membrane.
Other important information had been revealed by the crystal structure of the shortened dynamin. It appeared that the protein contained three amino acids that were critical for its GTPase activity. Also, that they were conserved among all GTPases with similar functions, providing a ‘signature’ for this group of enzymes. New questions, of course arise upon which the Schmid team is investigating. Schmid confessed that most of the questions are yet to be answered by the structure.
The research was published on April 28, 2010, in an advance, online issue of the prestigious journal Nature.