Three Definitely Isn't a Crowd When It Comes to Protein Complex Analysis

In a study published in Cell Reports, members of the Washburn Lab described their three-pronged approach to analyzing protein complexes – affinity tag protein purification, chemical crosslinking with high-resolution mass spectrometry and computational molecular modeling with protein docking.

A variety of advanced technologies exist for proteomic analysis that have propelled the research space forward over recent years – but they are not without their individual limitations. Could combining a variety of methods be critical for overcoming some of the key challenges in the proteomics field?

Describing the latest work, Mike Washburn, Director of the Stowers Proteomics Center, said: "I haven't been this excited about new capabilities in a long time."

Technology Networks interviewed Washburn to learn more about the study, the individual capabilities of each technique adopted and how they can be combined to deepen our understanding of protein complexes.

MC: In this study, you combined three individual techniques to study protein complexes. Can you please expand on each of these techniques, and why you decided to combine them?

Mike Washburn: Affinity purification using the Halo tag - the Halo tag is a modified bacterial enzyme – originally a haloalkane dehalogenase. This small Halo protein becomes covalently attached to magnetic beads, allowing for efficient affinity purification of protein complexes. This is important for generating a good sample for further analysis using additional methods.

Cross linking and high-resolution mass spectrometry (XL-MS) - We crosslink subunits of a protein complex using the crosslinker DSSO, capturing positional information. After capturing this information, we identify the crosslinks by first denaturing the proteins, digesting them into pairs of crosslinked peptides, and then analyzing these molecules by mass spectrometry in an Orbitrap-Fusion Lumos mass spectrometer.  The Orbitrap-Fusion Lumos has important capabilities to efficiently and accurately analyze protein samples treated with DSSO, which has been an important advance in cross linking mass spectrometry technology.

In silico protein docking - If we can obtain a sufficient number of crosslinks between individual subunits for which structures are available, we can then use the docking platform HADDOCK to find energetically favorable positions of the subunits relative to each other within the limitations imposed by the length of the DSSO crosslinker.

Affinity purification is a critical step in the process to generate an excellent sample prior to analysis. The Halo tag system is a very good system to do this. Next, we decided to combine XL-MS with protein docking based on earlier work by many other groups. In particular, we referred to a guide by Orban-Nemath et al. published in 2018 in Nature Protocols, titled: Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data.

MC: In the press release you said, "the capabilities have all existed, and have been used together a bit, but not in great numbers" – why do you think this is?

MW: The Halo affinity purification technology that we use was developed prior to 2007 and marketed by Promega. The DSSO crosslinker used for XL-MS studies was developed/published in 2011. The DSSO crosslinker was cleverly designed to break apart in a mass spectrometer, which greatly aids the detection and identification of cross-linked peptides when analyzed in a particular type of mass spectrometer. The Orbitrap Fusion Lumos is such a mass spectrometer and was introduced in 2015. In addition to being highly sensitive and fast, the Fusion Lumos has the ability to take advantage of the mass spectrometry cleavable feature of DSSO. This greatly improves the detection and identification of cross-linked peptides from a complex biological sample.   

The HADDOCK protein docking tool has been under development since 2003. Protein structure determination has been ongoing with new structures being determined all the time.

All these platforms and technologies have more recently become advanced to reliably detect a sufficient number of protein crosslinks that map to enough structures determined for a protein complex to allow docking. Without a sufficient number of crosslinks, or a sufficient number of crystal/NMR structures for subunits of a complex, the docking is not possible.

MC: Why it is important to deepen our understanding of protein complexes? What applications might this have?

MW: Protein complexes are molecular machines. Understanding how the parts of these machines are assembled helps us to understand how the parts function together. We are then in a better position to understand what is happening at a functional level when improper versions of these machines cause human disease.

MC: Can you tell us more about the Sin3/HDAC protein complex? Why was it a focus in this study?

MW: Sin3/HDAC complexes prevent subsets of genes from being expressed (gene silencing). They function by deacetylating histones, causing DNA to become wrapped tightly around nucleosomes and preventing gene transcription. The main scaffolding protein for the complex, SIN3A, is mutated in several human cancers, suggesting that aberrant function of the complex has a role in disease.

MC: Can you please discuss your key findings from the study, and how you plan to progress the research?

MW: We have found that it is possible to use the crosslinking data to dock Sin3 subunits together. For a deeper understanding of complex assembly and function, we need to dock more subunits and refine our models. To do this we need more detailed crosslinking data. Currently we can only crosslink unmodified lysine residues. If we can develop crosslinkers that will crosslink other residues, we will be able to fill in the gaps, get more detailed positional information, dock more subunits and get a more sophisticated picture of complex architecture and function.

MC: In the press release you said, "I haven't been this excited about new capabilities in a long time." – Please can you expand on this? What challenges have existed in this field thus far, and what doors have potentially been opened by combining these three methods?

MW: The structure of many protein complexes are yet to be elucidated using existing structural biology technologies. Having the ability to build integrated structural models of protein complexes using biochemical, mass spectrometry and computational approaches is powerful and broadly applicable and could help advance the field of structural biology.  In the future, we are particularly interested in studying how protein complexes change under different cellular conditions, or when mutations are introduced, for example.  We can use the approaches described in our current body of work to carry out such studies of protein complex dynamics. 

Mike Washburn was speaking to Molly Campbell, Science Writer, Technology Networks.

Reference: Banks et al. (2020). Integrative Modeling of a Sin3/HDAC Complex Sub-structure. Cell Reports. DOI: https://doi.org/10.1016/j.celrep.2020.03.080.