Complex architecture of "Molecular Giant" understood in greater detail than ever before

Complex architecture of “Molecular Giant” understood in greater detail than ever before

The molecular giant and its missing screws

The human nuclear pore complex (NPC) is a true molecular giant, located on the membrane that separates the nucleus from the cytoplasm. It is monk-shaped and acts as both a gateway and a control point for molecules that travel between the cytoplasm and the nucleus. Thereby, NPC facilitates basic processes in the cell, such as gene expression and translation. The nuclear transport system also plays a role in several diseases, including neurodegenerative disorders, cancer and viral infections.

What is the structure of the NPC? How is its protein glued together? How does it attach to the core membrane? These and other questions have now been answered by Kosinski Group at EMBL Hamburg and Center for Structural Systems Biology (CSSB)the Hint and Lobster Labs at Max Planck Institute of Biophysics and staff. They created the most complete model of the human NPC to date by combining the AlphaFold2 protein structure prediction program with techniques such as cryoelectron tomography, single particle cryo-EM and integrative modeling.

For structural biologists, the human NPC is a challenging but exciting 3D puzzle, with about 30 different proteins, each of which is available in several copies. It is about 1000 pieces of the puzzle, which form a round core with surrounding flexible parts. Until now, the most accurate models of the human NPC core covered only 46% of the structure. But now, based on two decades of previous research in the field, researchers have created a new model of the NPC structure that covers more than 90% of its core.

While previously proposed NPC models had gaps and contained certain proteins only in fragments, the new model removes much of this ambiguity.

“It’s like disassembling and assembling an electronic device. There will always be some screws left, and you just do not know where they should be, says EMBL’s group leader Jan Kosinski, who led the investigation. “We finally managed to fit most of them, and now we know exactly where they are, what they do and how.”

Experiment and artificial intelligence work together

How did the researchers achieve this? The key was to combine several experimental and computational methods. This allowed the researchers to visualize NPCs at different scales and levels of detail.

For example, to model the overall silhouette of the NPC, the researchers used cryoelectron tomography. With this technology, they were able to observe NPCs in its cellular environment, rather than in isolation. More details about the individual protein building blocks were revealed by AlphaFold2an artificial intelligence-based program that predicts protein structures, created by the company DeepMind.

“AlphaFold2 was a breakthrough moment for us,” said Agnieszka Obarska-Kosińska, a postdoc who performed the molecular modeling. “Previously, we did not know the structure of many proteins in NPCs. You can not solve a puzzle when you do not know what the pieces look like. But AlphaFold2 combined with other approaches made it possible for us to predict these forms.”

To further refine the picture, the researchers used ColabFold, a version of AlphaFold2 modified by the research world to model interactions between proteins. This made it possible for them to visualize how the different pieces of the puzzle are combined to form smaller sub-complexes, and how these sub-complexes are then glued together to form NPCs.

Finally, they put all the parts together using the software Mounting previously developed by Kosinski Groupand validated it against experimental data.

The resulting model was so complete and detailed that it allowed researchers to create time-resolved molecular simulations that explain how NPCs and the nuclear membrane interact to create a stable pore and how it responds to mechanical signals.

Future orientations

This work was a major step forward for NPC research, but there is still much to explore.

“This work exemplifies how structural biology in the future will include cell biology to create atomic models of ever-increasing assemblies of molecules that perform different functions in different parts of the cell,” said Martin Beck. Gerhard Hummer agrees: “We can now imagine building a complete dynamic model of NPCs and simulating nuclear power transport in atomic detail.”

The Kosinski Group will focus its future work on developing automated methods for integrating structural and microscopic data using AlphaFold2 and their own software. Mounting. They plan to apply these methods to study molecular processes that drive viral infections.

Studying NPCs in their cellular environment is also in line with the objectives of the new EMBL program ‘Molecules for ecosystems‘, which aims to explore life in its context.

Reference: Mosalaganti S, Obarska-Kosinska A, Siggel M, et al. AI-based structural prediction enables integrative structural analysis of human nuclear pores. Science. 2022; 376 (6598): eabm9506. doi: 10.1126 / science.abm9506

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