For\u00a0nearly\u00a0half\u00a0a century, scientists have known that malaria parasites force their way into human red blood cells through a ring-shaped structure called the moving junction. What no one could work out was what it\u00a0actually does. The structure assembles, does its job, and\u00a0dissipates\u00a0in the space of 60 seconds\u00a0\u2013 gone before anyone can get a close look.\u00a0<\/p>\n
A team at Columbia University has\u00a0now finally\u00a0caught the moving junction in the act. By freezing parasites at the\u00a0onset\u00a0of invasion and lifting the intact complex straight out of the cell, the researchers obtained the first high-resolution view of its three-dimensional structure. What they saw overturned a decades-old assumption about how the parasite gets in.\u00a0Rather than\u00a0a passive doorway, the moving junction turns out to be a molecular machine that actively remodels the host cell’s membrane to help the parasite force its way inside.\u00a0<\/p>\n
The findings, published today in Cell, detail how the team obtained the structure and then used it\u00a0as a blueprint\u00a0to\u00a0designed\u00a0a mini-protein, from scratch, that blocks invasion-a proof of concept for a new kind of antimalarial drug.\u00a0<\/p>\n
Malaria still kills\u00a0roughly\u00a0600,000\u00a0people\u00a0a year,\u00a0the overwhelming majority of\u00a0them\u00a0young children\u00a0in sub-Saharan Africa, and the parasite is steadily becoming resistant to frontline drugs.\u00a0The\u00a0disease\u00a0starts with\u00a0a single event: a parasite breaking into a red blood cell.\u00a0In an infected person,\u00a0trillions of parasites are released and invade every\u00a048 hours\u00a0in synchronized waves. This rhythmic cycle of rupture and reinvasion drives the periodic\u00a0fevers malaria is known for.\u00a0The\u00a0same\u00a0moving junction\u00a0machinery is\u00a0used\u00a0across every species and every stage of the\u00a0parasite’s\u00a0life cycle, which has made it one of the most sought-after targets in malaria research. Block it, and you\u00a0stop\u00a0infection at its source.\u00a0<\/p>\n
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We’ve known for decades that this structure is essential for the parasite to get into a cell, but not how it actually works. Pulling it directly out of the parasite intact let us finally ask that question directly.”<\/p>\n
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Chi-Min Ho, Assistant Professor in the Department of Microbiology and Immunology at Columbia University Vagelos College of Physicians and Surgeons and study’s senior author\u00a0<\/p>\n
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Catching\u00a0parasites in the act\u00a0<\/p>\n
The moving junction has been a puzzle since 1978, when scientists first\u00a0observed\u00a0a mysterious thickening of the membrane where parasite meets\u00a0cell\u00a0in electron microscopy images. Researchers eventually\u00a0identified\u00a0the four parasite proteins that assemble into the junction’s basic building block-known as AMA1, RON2, RON4 and RON5-and confirmed that all were essential for invasion. But what the structure\u00a0actually did\u00a0remained\u00a0unknown, because\u00a0it survives for less than 60\u00a0seconds and refuses to reassemble in a test tube.\u00a0<\/p>\n
The Columbia team got around this by stopping invasion mid-stride. Using a compound that halts the parasite’s internal motor without preventing the junction from forming, they stalled parasites partway into red blood cells, then extracted the fully assembled\u00a0AMA1-RON complex-the\u00a0building block from which the\u00a0whole junction is constructed-and\u00a0imaged it with cryo-electron microscopy, a technique\u00a0where\u00a0molecules\u00a0are flash-frozen and imaged with an electron beam at extremely high magnifications\u00a0to\u00a0reveal their shape in atomic detail. The result was a sharp, three-dimensional view of that building block. The researchers noted that it was\u00a0quite\u00a0strikingly\u00a0shaped\u00a0like a sailboat, with the AMA1 protein forming a “sail” above the cell surface and the three RON proteins forming a broad “hull” pressed against the membrane below.\u00a0<\/p>\n
Not a passive doorway at all\u00a0<\/p>\n
The biggest surprise was in the hull, where the team found clues that finally hinted at the moving junction’s role in invasion.\u00a0The face of the structure pressed against the host membrane is blanketed\u00a0with positively charged anchors, and the surface is\u00a0studded with short helices that drive deep\u00a0into the membrane like wedges. Both features are\u00a0widely\u00a0recognized\u00a0hallmarks of a\u00a0well-known\u00a0family of cellular machines that bend and reshape membranes.\u00a0<\/p>\n
To test whether the structure could\u00a0indeed\u00a0deform a membrane, the researchers synthesized the parasite’s wedge-like helices and added them to artificial membrane bubbles. The\u00a0membranes thinned and punctured. Meanwhile,\u00a0weakened versions of the helices left\u00a0the bubbles intact. The team concluded that the moving junction\u00a0appears to pull\u00a0the host membrane into shape,\u00a0likely working\u00a0in concert with the\u00a0parasite’s\u00a0motor to lever the parasite inside.\u00a0<\/p>\n
“It had been pictured as a kind of series of staples or spot-welds, making up a passive ring the parasite hauls itself through,”\u00a0said Meseret Haile, the study’s first author and a PhD candidate in Ho’s lab.\u00a0“What we see instead is a machine built to reshape the host cell’s own membrane. That changes how we think about the whole event.”\u00a0<\/p>\n
A blueprint for new drugs\u00a0<\/p>\n
Beyond\u00a0finally revealing\u00a0how the moving junction allows the parasite to invade, the structure also\u00a0gave\u00a0the team a precise map of where and how AMA1 grips its partner protein, the contact that holds the entire junction together. Using a machine learning-poweredprotein-design tool together with their structural information, the researchers designed a\u00a0mini-protein to break that grip. Their best candidate blocked parasites from invading red blood cells in a dose-dependent way and left already-infected cells unaffected, confirming that it works\u00a0specifically\u00a0by stopping entry rather than through general toxicity.\u00a0<\/p>\n
The designed\u00a0mini-protein\u00a0is a first proof of concept, not a drug, and will need considerable refinement before it could be tested in people. But it\u00a0demonstrates\u00a0an exciting new\u00a0strategy: using near-native structures to design invasion-blocking mini-proteins\u00a0against a target that has long frustrated conventional approaches. The same structure also clarifies how several leading anti-malaria antibodies work, information that could feed back into vaccine design.\u00a0<\/p>\n
“Once we could see the target in its real setting, designing something to block it became a tractable problem,” said Daphne Kaxiras, an MD-PhD student in Ho’s lab who led the inhibitor design. “That’s the part we’re most eager to build on.”\u00a0<\/p>\n
The team’s approach,\u00a0imaging\u00a0fragile complexes\u00a0captured\u00a0directly from the organism and using them to guide design,\u00a0may apply to many other parasites and pathogens\u00a0that are notoriously difficult to study.\u00a0<\/p>\n
Source:<\/p>\n
Columbia University Irving Medical Center<\/a><\/p>\n Journal reference:<\/p>\n