Scientists have cracked the structure of a protein that is vital to the parasite Plasmodium falciparum, the one that causes the most deadly form of malaria. They suggest the protein, a key enzyme in the generation of cell membranes, could be an ideal target for anti-malaria drugs, particularly as the protein is not present in humans.

The study was led by the Department of Biology at Washington University, St. Louis, Missouri, and a report on it appears as the “Paper of the Week” in the 6 January issue of The Journal of Biological Chemistry.

In 2010, malaria killed 655,000 people worldwide. The disease is caused by five different species of Plasmodium, a parasite that lives in the gut of its primary host, the mosquito, but the deadliest form of malaria comes from being bitten by a mosquito carrying the species Plasmodium falciparum.

New drugs to combat malaria are desperately needed: not only is P. falciparum responsible for the most severe form of malaria, it is endemic in areas populated by about 40% of the people in the world, and drugs that used to work are losing their effectiveness, partly because counterfeiting has led to widespread resistance.

In a biology lab at Washington University, researchers took six years and more to uncover the structure and function of the protein, an enzyme called PMT (short for phosphoethanolamine methyltransferase).

In previous work they had already established that the enzyme’s job is to add methyl groups to a starting molecule called phosophoethanolamine that is involved in making the cell membranes.

And even though there are similar proteins in other organisms, humans don’t have it.

These features make it an ideal target for developing new anti-malaria drugs.

Senior author Dr Joseph M. Jez, associate professor of biology in Arts & Sciences, told the press:

“What my lab does is crystallize proteins so that we can see what they look like in three dimensions.”

“The idea is that if we know a protein’s structure, it will be easier to design chemicals that would target the protein’s active site and shut it down,” he added.

The researchers have perfected an interesting way to crystallize a protein. They put a solution of a salt or something else that can dry out the protein at the bottom of a small well. Then, as Jez explains: “we put a drop of our liquid protein on a microscope cover slip and flip it over the top of the well, so the drop of protein is hanging upside down in the well”.

This helps to slowly withdraw water from the protein, rather like making rock candy, except in the case of candy it’s the string hanging into the jar of sugar solution that helps to withdraw water.

There is also another difference: in making rock candy, the sugar is not reluctant to form crystals, but in this process, the protein is highly reluctant.

In fact, it took six years of painstanking research for them to screen 8 proteins through a total of 4,000 conditions. They used 24 wells to a tray, at a rate of around 500 wells per protein. And then they also had to try different combinations of ligands to the proteins and crystallize those as well.

Most of this work was done by first author Soon Goo Lee, a doctoral candidate in Jez’s lab.

At this point you might ask, why do you need to make a crystalline form of the protein to determine its molecular structure in 3D? That becomes clearer when you realize that a really nice big crystal produces a strong, clear scatter pattern when X-rays are projected through it. The pattern is produced by the distinct line-up of atoms in the crystal, which is never the same for different molecules.

While the scatter pattern of the X-rays itself does not reveal the 3D molecular structure of the protein, with a good, clear X-ray scatter pattern, the scientists have enough mathematical information to back-calculate the relative positions of the atoms in the protein molecule.

It’s a bit like throwing a bunch of pebbles into a still pond and then using the pattern of waves arriving at the edges to work out where the pebbles went in.

While this metaphoric description makes it sound easy and straightforward, in practice it is very hard. There were lots of technical difficulties with getting the Plasmodium enzyme finally to crystallize, including the fact when it did, four paper-thin crystals emerged, stacked on top of each other.

Jez explained that when they took the block of crystals to be X-rayed, Lee “actually did surgery under the microscope and cracked off a little tiny piece of it”, and to everyone’s surprise, they managed to get a clean diffraction pattern.

The moment of truth came when they put the diffraction results into the computer, did the back-calculation, and Lee paused with a finger ready on the mouse button: a final click would reveal whether the years of hard work had paid off.

They had. When Lee clicked the mouse he saw a clear electron density map in exceptionally sharp focus.

Jez explained that the next step was to use the electron density map to build a structure that matches the amino acid sequence of the protein:

“The first thing you do is put in the amino acid backbones and connect them together to form a chain. It’s like having a long thread, each inch of which is an amino acid, and your job is to take that thread and move it in three dimensions through that electron density map.”

Then you have to add the side chains that make one amino acid different from another.

“The amino acid sequence is known,” said Jez, “Your goal is to match the way you string together the amino acids in the electron density map to that sequence.”

The researchers created a “cartoon” of the electron density map to make it easier to see the protein’s structure and figure out how it works.

The cartoon helped them “see” how the molecules involved are positioned in the active site of the enzyme, the “pocket” if you like, where the chemistry takes place.

Jez said:

“The PMT enzyme is trying to join two molecules. To do that, it has to lock them in place so that the chemistry can happen, and then it has to let go of them.”

He said they think the protein has a “lid” that opens and closes: it stays open, leaving the site active, until the substrates enter, and it clamps shut, and when it does this, it puts the substrates together.

The researchers note in their report that while these insights into the structural characteristics and functions of PMT are beginning to reveal some potential targets for anti-malaria drugs that may kill Plasmodium without harming humans, they say further studies are now needed to “understand the evolutionary division of metabolic function in the phosphobase pathway”.

It may be a significant step, but there’s still a long way to go.

Written by Catharine Paddock PhD