McArdle Blog ~ 10/8/2014

To know what it does you must know how it’s built:
The structure function relationship in biology

Imagine you are hard at work trying to solve a jigsaw puzzle and things have started quite well. You are chugging along merrily and have about a third of the picture reconstructed. Then you run into a peculiar problem: you start finding pieces whose edges are covered with duct tape. Without being able to see how these pieces – with hidden edges – fit with  each other or the other pieces you cannot complete the puzzle.
Yongna Xing

Structural biologists like Dr. Yongna Xing at the McArdle Laboratory for Cancer Research tackle similar problems in the field of molecular biology. Often scientists will know two proteins are part of the same picture, or complex, within our cells but they won’t know the details of how these two proteins are interacting with each other.

But understanding the structural basis, the nuts and bolts if you will, of how proteins are interacting with each other in our cells is crucial if we are complete the puzzle of how they function.

In a paper published on September 25th  in the journal Cell Reports Dr. Xing and her colleagues report the structure of two proteins, MTDH and SND1, interacting with each other, and how this interaction is necessary for the cancer-promoting activities of these proteins.

THE DEADLY DUO

MTDH, or metadherin, is often present at higher levels in cancer cells than in normal cells. In fact, Dr. Xing says “MTDH is overexpressed in almost every kind of cancer.

In tumors MTDH is involved in several processes such as boosting cancer cell growth, preventing death of cancer cells, promoting metastasis – the movement of cancer cells from the original tumor site to other parts of the body – and increasing blood and nutrient supply to tumors. But exactly how MTDH performs all these functions has remained frustratingly unclear.

One way to find out more about how a protein performs its functions is by searching for other proteins it interacts with; MTDH interacts with a slew of other proteins, including one called SND1.

Why focus on SND1 if MTDH interacts with many other proteins? “Because these two proteins [MTDH and SND1] are both overexpressed in multiple types of cancer you probably would think their direct interaction would play a role [in cancers]” says Dr. Xing.

The first clue that MTDH-SND1 interactions play important roles in cancer cells was that MTDH mutants unable to interact with SND1 reduced its ability to enhance tumor cell growth, survival and metastasis.

But “It’s not sufficient to prove that  this interaction is relevant in dictating function in cancers”, says Dr. Xing, “and that’s where we need to know how the two proteins interact at atomic level.

LOOKING DOWN THE ATOMIC HOLE

In order to begin to understand how interactions between MTDH and SND1 contribute to so many cancers Dr. Xing’s lab wanted to get a snapshot of what the MTDH-SND1 interface actually looked like. But proteins are tiny molecules, so small that even peering through powerful microscopes would not allow us to get a picture of their structure.

The Xing lab used a process called X-ray crystallography to determine the structure of the MTDH-SND1 interaction surface at atomic level, which gives a resolution in Angstroms - 100,000-fold smaller than the size of mammalian cells.

Explaining exactly how X-ray crystallography works is beyond the scope of this blog, but the National Institute of Health has a fantastic description of the process here and there is a great two-part video from the Royal Society here.  

Dr. Xing and her colleagues had to hurdle several obstacles while using this complex process. For example, one of the key steps in determining a protein structure using X-ray crystallography is to produce crystals of the protein. The more regular the crystal is and fewer the number of imperfections, the greater clarity and detail of the final structure.

The Xing lab had to generate a crystal of TWO proteins interacting with each other. But even “after extensive effort, cocrystallization…failed to yield protein crystals”, probably because the bond between MTDH and SND1 isn’t strong enough to stabilize the complex for crystal formation; no crystals = no structure.

But experimental failures are a daily occurrence in the life of all scientists, who learn to roll with the punches and keep trying. The Xing lab didn’t give up ; “I think we succeeded because we tried some unique approaches” says Dr. Xing.

The Xing lab hit upon the idea to hold the MTDH and SND1 proteins close to each other by essentially tying them together. Now that the proteins were physically close to each other, Dr. Xing and her colleagues could generate the crystals. Even then “to go from when we got the first crystal to finishing data analyses and the first structure took over a year” explains Dr. Xing.

GOING BEYOND THE STRUCTURE…INTO FUNCTION

The structure of the MTDH-SND1 complex gave Dr. Xing important insight into what the SND1 and MTDH interface looked like. Armed with knowledge of how these protein jigsaw puzzle pieces fit together they proceeded to alter their structures at the interface surface.

MTDH-SND1Proteins are made up of different combinations of molecules called amino acids much like how houses can be made up of combinations of various building materials. Just as how a house made of bricks and concrete will be quite different from one made of logs and thatch (but they will both still be houses), proteins can have widely different structures and functions depending upon what combination of amino acids they are made of.

Knowing which amino acids are present at the interface of MTDH and SND1 allowed the Xing lab to swap out specific amino acids for others and test whether these substitutions affected the MTDH-SND1 interactions.

Dr. Xing and her colleagues found the amino acids they had predicted to be important for MTDH-SND1 interactions based on the structure they had derived were in fact vital for the proteins to interact.

Using a technique called Co-IP the Xing lab was able to show that if they changed specific amino acids in either MTDH or SND1 they could no longer interact with each other. Dr. Xing now had the beginnings of a map showing exactly how MTDH and SND1 formed a complex together.

But how important is this bond between MTDH and SND1 for the ability of these proteins to promote various cancers? Knowing exactly which amino acids can be modified to abolish the interaction between the two proteins allowed the Xing lab and collaborators to test exactly that question.

The data proved our prediction that this interaction might be important for the function of both MTDH and SND1 and their roles in cancer promotion”, says Dr. Xing. “The surprising part is how robustly this interaction affects cancer promotion”.

The Xing lab and collaborators found that if altered MTDH or SND1 unable to bind its partner-in-crime was used, tumors were much smaller and showed slower rates for growth and formation. The link between structure and function had been established.

WHERE DOES THE FUTURE HOLD?

When I ask Dr. Xing where she sees this research going she gets a far-away look in her eyes, but answers without hesitation. “Although we know this interaction is crucial for cancer promotion, what are the specific steps downstream of this interaction?” she asks. The Xing lab will try to answer this question and they already have a few ideas.

Dr. Xing explains that “MTDH can interact with multiple binding partners…and it seems almost like the MTDH-SND1 complex serves as a bridge to allow the interaction of multiple signaling molecules in the same complex, which eventually leads to cancer promotion.

Ultimately “This interface can potentially be a therapeutic target” says Dr. Xing. Their structural studies have identified “two interacting pockets in SND1 that can be targets for developing therapeutics” and the Xing lab is now gearing up to test different chemical compounds that can inhibit the interactions between SND1 and MTDH.

 The structural studies completed by the Xing lab allows them to design experiments to determine what proteins are part of the MTDH-SND1 complex and how disrupting this interaction changes the profile of other proteins in this complex.

Knowing which proteins are brought together by the MTDH-SND1 protein bridge and what cellular processes is affected by this interaction may just help us understand how this complex promotes so many different tumor types and how we can go ahead and target this complex for various cancers.

 

~Rup Chakravorty