Another Eukaryotic Gene Regulator Turns up in Archaea

Leucine zipper proteins as gene regulators in Archaea, regulated by nutrient-binding PAS domains.

In this day of AI, it is a pleasure to read an elegantly constructed and written paper that breaks new ground in biology. A group in Berkeley has discovered that a common gene regulatory family in eukaryotes, the leucine zipper proteins, have clear antecedents that were impossible to detect with pure sequence comparisons, but which turned up during their work on the nutritional regulation in archaeal methanogens. They note that archaea are now well-known to be the progenitors of the nucleus and nuclear functions of eukaryotes, (as well as translation, the cytoskeleton and much else), but that there has been a shortage of known regulators to account for the complex lifestyles of archaeal cells, particularly these methanogens.

Methanogens, as one might guess, give off methane, creating small amounts of the natural gas we take to be a mineral resource. For them, methane is an electron dump, (instead of oxygen, as it is for us), from food sources such as methanol, methylamine, and many other methylated compounds, which are slightly digested to form CO2 and methane. Methanogens are anaerobic, meaning they are poisoned by oxygen and originated from the early epochs of earth history before there was much free oxygen. It is naturally critically important for these organisms to know what kind of food is out there, since digestion of methylamine calls for different enzymes than the digestion of methanol. The authors thus had the hypothesis that there should be some regulatory apparatus for sensing methylamine (in this case) and activating the right genes to digest it. 

These authors performed a laborious laboratory evolution experiment, looking for mutant bacteria which were able to switch between food sources faster than the wild type Methanosarcina acetivorans, which take about four days to switch from methanol to trimethylamine. There were replicate populations, repeated passages of these slow-growing bacteria to new cultures with different food sources, (all under anaerobic conditions), and sequencing of the resulting strains. And then CRISPER-based specific mutagenesis to confirm the mutations that caused the phenotype of more rapid food switching. In the end, they obtained strains that took only 17 hours to switch food sources- much faster than the wild-type. The most common gene that was mutated was MA2561, which is a repressor of a methyltransferase specific to trimethylamine. These mutations disable the repressor and allow this gene to be expressed all the time, which would be expensive for the archaea in the wild, but in the lab, dramatically shortens the time needed to acclimate to the new food source. They renamed this gene amzR, for amazing regulator!, er, actually ... "archaeal metabolite-sensing zipper-like regulator".

The interesting thing is that this regulator and its family had not been recognized in archaea or in this species previously, even with all the computational power available to find homologs of any kind of protein. But once it was found by this functional screen, and the authors paid some attention to it, and put it through the alpha-fold program, then everything became clear. It is structurally similar to a large family of eukaryotic DNA-binding gene regulators called leucine zipper or b-zip proteins. This is because they contain a dimer of stiff alpha helices that wind around each other (using hydrophobic leucines at key contact positions) and make a scissor-like fork whose (basic) end binds to DNA. 

Comparison of leucine zipper structures. The authors provide an overview of structures. A is a garden-variety eukarotic b-zip protein, (GCN4), a regulator of amino acid biosynthetic genes. B is a more complex basic semi-zipper protein that combines DNA binding with PAS domains that are, in this case, sensors for oxygen levels, with significant psychiatric implications. C shows the newly found amzR repressor, in full dimeric mode with its PAS and GAF domains, which appear to sense trimethylamine. Lastly, D shows a helix-turn-helix DNA regulator that is characteristic of bacteria, though common in archaea and eukaryotes as well.

In addition, amzR has a couple of other domains- a PAS domain and a GAF domain, which are similar to each other, and are common motifs that bind small molecules, in this case likely the trimethylamine that causes the whole chain of events. The authors show that amzR binds DNA, that it represses the MT1 gene that allows growth in trimethylamine, and that it binds to trimethylamine itself, which impairs DNA-binding, presumably through structural shifts in dimerization or the orientation of the dimer. Thus, it is what is quite common in archaea and bacteria- the one-component-regulator which both senses and effectuates some kind of control in response to environmental conditions. 

They also find, using the new sequence, that direct homologs of amzR are common among methanogen archaea, and speculate that more distant relatives may account for some fair portion of the missing regulatory capacity that was remarked upon above. Unfortunately, given the inability of sequence comparisons alone to find these homologs, given the deep divergence in time among all these organisms, it remains difficult to speculate just how widespread this regulatory motif is in methanogens, or in archaea generally.

A somewhat indirect, but effective, demonstration of ligand binding. The amzR protein melts at high temperature, here tracked by its fluorescence. The melting temperature shifts significantly upward if a (presumably stabilizing) ligand is offered to bind. NS = no substrate; MMA = monomethylamine; DMA = dimethylamine; TMA = trimethylamine.

The authors offer a basic model of control, where amzR (aka MA2561) binds to and represses the multi-gene operons controlling trimethylamine digesting methyltransferases among others. The inducing chemical, TMA, binds to and inactivates this repressor, turning on the operons.

• Thoughts on retirement savings.