Thermus aquaticus and T-Rex

By: Alison Wong and Elizabeth Speyer Besancon


Ah, the hot springs. Relaxing, warm and wonderful, hot springs are the perfect place to go for vacation (and in just about every ‘gratuitous vacation’ in Japanese anime).

This is a hot spring in Yellowstone National Park, a place too hot for most

organisms but perfect for Thermus aquaticus. Source

Doesn’t that just look wonderful?

Granted, I’ve never been to a hot spring, but I sure wish I could go to one. I’d say just about all microbes would disagree with me, citing reason: death. The exception is the community of thermophiles that love the heat, for example, the thermophilic bacteria Thermus aquaticus.

Thermus aquaticus. Source

Thermus aquaticus loves the heat (specifically, 70 - 75 degrees Celcius), and lives happily where most bacteria would die--such as in the hot springs of Yellowstone, pictured above, and in other extremely hot water.  If T.  aquaticus were a video-game character, its heat-resistance level would be ridiculously high.

Since T. aquaticus is extremely heat resistant, it stands to reason that the proteins T. aquaticus uses for daily living  are also heat resistant, such as its famous heat resistant DNA polymerase, (nicknamed Taq Polymerase) and plenty of other enzymes. Taq polymerase has quite a lot of uses and applications- the Polymerase Chain Reaction was able to be automated through the use of T. aquaticus’s  (where other DNA polymerases would denature), and automated PCR is used in a myriad of things, such as identification of disease and DNA sequencing.

Here we take a look at another such heat-resistant protein belonging to T. aquaticus, this time, involved in regulating its respiration and metabolism. This protein, T. aquaticus’s Rex protein, regulates the genes for respiration and changes shape in response to the NADH/NAD+ ratio. Its structure had  been quite the mystery until recently.

The Metabolic Strategy of T. aquaticus, and the Unnecessity of Oxygen

For many organisms, oxygen is critical for life. For others, oxygen is toxic. But for some special microbes like Thermus aquaticus, oxygen is optional. T. aquaticus is able to switch between aerobic respiration, the usual metabolic strategy for organisms that breathe air, and fermentation, a common metabolic strategy for organisms that live where oxygen is scarce.

Fermentation uses energy inefficiently, so it is better to avoid it when oxygen is around. Respiration is much more efficient at extracting energy from sugar but it won’t work without oxygen. Therefore, T. aquaticus needs a way to distinguish between high and low oxygen environments. To do this, Thermus aquaticus’s Rex protein monitors the ratio of NADH to NAD+, which goes up in the absence of oxygen.

Protein Structure and Function

Source

The purpose of the experiment performed by Krystle McLaughlin and her colleagues was to find the structure and function of Thermus aquaticus’s Rex protein--T-Rex for short (no relation to the dinosaur shown to the right).

Before this experiment took place, a few things about T-Rex were already known. It is a DNA binding protein that controls genes involved in respiration, like other Rex proteins. It has a dimer structure (meaning that two copies of T-Rex, called subunits, bind together and work as one unit; just as a pair of pants can’t work as an individual pant, nor can a single copy of T-Rex function alone). It binds to NADH and the presence or absence of NADH to determine whether or not T-Rex binds to DNA. All else was unknown until recently.

Through X-ray diffractions and computer modeling, McLaughlin and her team figured out both the structure of T-Rex and how it works. In x-ray diffraction, a protein (or other molecule) is isolated and crystallized. Then, an x-ray beam is shined on it and the pattern of light diffraction is recorded, which gives an image of the electron density in different parts of the crystal. By repeating this with the crystal positioned at different angles and running the data through computer algorithms, every atom can be located.

Each subunit of the T-Rex dimer has two binding sites: a DNA binding site near the end called the N-terminus and an NADH/NAD+ binding site near the other end, the C-terminus. When there is lots of oxygen in the cell, a molecule of NAD+ is bound to the NADH/NAD+ binding sites of the two subunits. Every so often, the NAD+ will be knocked out of the binding site, allowing NAD+ or NADH to bind. As long as the cell has plenty of oxygen, the number of NAD+ molecules will outnumber the NADH molecules, so another NAD+ molecule will bind.

As long as the NADH/NAD+ binding site is open or bound with NAD+, the DNA binding sites in the two subunits will be aligned in a way that allows the T-Rex dimer to bind with a specific segment of DNA. This DNA comes right at the start of a set of genes that would start fermentation. T-Rex occupies the space where RNA polymerase would bind to start transcribing genes. Since RNA polymerase can’t bind, the genes for fermentation don’t get transcribed into RNA, no RNA means no proteins, and no proteins means no fermentation. This is good for the cell because it doesn’t want to ferment when there is plenty of oxygen available. Fermentation is inefficient at getting energy from sugar.

However, when oxygen is low, the cell needs to ferment. Without oxygen, NAD+ is used up and NADH accumulates. When NAD+ is knocked out of the NADH/NAD+ binding site, it is replaced with NADH instead of NAD+. When NADH binds, the whole T-Rex dimer changes shape. The T-Rex subunits rotate into a closed conformation that can’t bind to DNA. You can see this in a video here, or just look at the drawing below:

On the left, NAD+ is bound to T-Rex, allowing it to bind with DNA. When NADH replaces
NAD+, T-Rex adopts the closed conformation seen on the right, preventing DNA binding.
Y, D, and R represent regions that the scientists tracked to determine conformation. Source

With this closed conformation, T-Rex drifts away from the DNA, opening a space for RNA polymerase to bind. This allows the genes that perform fermentation to be transcribed and translated, so the cell is flooded with proteins for fermentation. This allows the cell to replenish its supply of NAD+ and continue living.

Paper Critiques and Looking to the Future

This paper did what it set out to do: it presented the scientific community with every detail they could possibly need to know about the structure of T-Rex. That’s what other scientists involved in finding protein structure needed to see to determine that their conclusions were sound. Since they were writing for people who would want to know everything, it was hard for the casual reader to understand what they were talking about. Hopefully, this summary has bridged that gap in understanding.

The main possibilities for future research lie in finding similar mechanisms in other organisms. The researchers hint that there are some eukaryotic species which may sense oxygen levels in a similar way, using a protein dimer to react to the ratio of NADPH to NADP+. Knowledge leads to more knowledge, and we can’t know what lies at the end of the path until we’re already there.



References

McLaughan, K.J., Strain-Damerell, C.M., Xie K., Brekasis D., Sorares A.S., Paget, M.S.B. and Kielkopf C.L. (2010).  Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor.  Molecular Cell, Volume 38. pp. 563-575.