Proteins usually work together, whether as part of a pathway or a complex, and there are several ways to investigate protein interactions. Scientists have now combined several of these approaches to learn more about the relationship between neighboring surfaces in a protein complex called Sin3/HDAC. The complex has been implicated in cancer, and is a cancer treatment target. The work, published in Cell Reports, applied chemical crosslinking, high-resolution mass spectrometry, affinity tag purification, and computational modeling, and can be used for the study of other complexes in the future.
"Putting all these pieces together gives us a new perspective on how protein complexes are put together, "with the potential to provide more information more quickly," explained Michael Washburn, Ph.D., director of the Stowers Proteomics Center. "The capabilities have all existed, and have been used together a bit, but not in great numbers yet. It's certainly complementary to existing techniques such as nuclear magnetic resonance (NMR), cryo-electron microscopy (EM), and X-ray crystallography, to really understand how protein complexes assemble, and to see how are they really interacting. What happens when you perturb them with drugs or mutations?"
The Sin3/HDAC complex moves the histone deacetylases (HDACs) to specific locations in the genome, where they suppress gene expression by taking acetyl groups off of histone tails. While the various parts of the Sin3/HDAC complex are known, obtaining information about the entire complex has been challenging.
Mass spectrometry can identify molecules in a mixture, but it provides no information about how they interact, noted the co-first study author Charles Banks, Ph.D. There are also methods to assess an interaction between two proteins, but they can be time-consuming, he added."What would be really good is if we could capture some sort of structural information about the intact complex before we separate the proteins, and that's where crosslinking comes in," Banks explained.
The researchers used a chemical crosslinker called disuccinimidyl sulfoxide (DSSO), winch crosslinks lysine residues in protein complexes that are within around 30 angstroms of each other. That provides spatial information about the complex.
"If I see a crosslink between two lysine residues, that means in the fully intact folded protein, those lysines are quite close together, giving us positional information," explained Banks. "And if I've got two different proteins, and two lysine residues - one from each protein - crosslinked together, that means those lysines in those two proteins are usually very close together, which is consistent with the proteins interacting with each other."
The study revealed 66 crosslinks between proteins, and 63 crosslinks inside 13 subunits of Sin3 , which revealed the relative positions of 5 of the Sin3 scaffold subunits: SAP30L, HDAC1, SUDS3, HDAC2, and ING1.
"We think of Sin3A as a platform on which all the other subunits assemble," explained Banks. "We still aren't sure which subunits can bind to Sin3A at the same time, although we know that some of them certainly don't bind at the same time."
"But why are there all these other subunits other than the HDACs? Why can't you just recruit the HDACs?" he asked. "We think that probably, the Sin3 complexes control transcription of a specific set of genes, maybe at a specific time and place in specific cells, and we think the other subunits are probably governing that specificity."
Molecular modeling with crosslinking information is "like laying the foundation for getting the complete structure of really large complexes, which is hard to do," said Washburn.
"This is a convergence of technologies that allows us to, going forward, do this on really any protein complex under almost any circumstance...You just have to be able to purify it. If you can get enough material, you can really study any protein complex. It's a matter of doing good biochemistry and preparing a good sample. If you can get really spectacular data, you can build models like this," Washburn added. "I haven't been this excited about new capabilities in a long time."