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How Cells Keep Right-Handed Amino Acids Out

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One of the wonders of life at the molecular scale — a fact that defies chance — is the purity of left-handed amino acids in proteins. Without this “homochirality,” as it is known, proteins would never fold properly into the functional structures that make life possible.

Theoretically, life could be built backwards, using only right-handed amino acids — as long as the mixture is pure. But the cell’s translation machinery (TM) would have to be redesigned to accommodate the change. (Note: left-handed amino acids are prefixed by L- and right-handed ones by D-).

Whenever amino acids form naturally, they occur in “racemic” mixtures of L- and D- “isoforms.” It’s been a long-standing mystery in the origin-of-life community how cells first discriminated between them. The physical and thermodynamic properties of L- and D- forms are identical; the only way they can be discriminated in the lab is by seeing which way they rotate polarized light. How could a primordial soup lead to a homochiral protocell? Unless the TM already existed to discriminate between the isoforms, the probability of getting a usable protein of any functional length is vanishingly small.

Exactly how the TM rejects the D-amino acids has been unknown. Now, a new paper in PNAS announces new findings about the discrimination process. Englander et al. from Columbia University and other institutions found two checkpoints that do not let D-isoforms pass. Especially interesting is an enzyme whose job is to take off D-amino acids that get put onto tRNAs by mistake by the family of aminoacyl-tRNA synthetases (aa-RS tRNAs).

Although the ribosome catalyzes protein synthesis using more than 20 chemically diverse natural amino acids, these natural amino acids are all of L- or achiral configurations. As a consequence, the ability of the ribosome to incorporate L-aminoacyl-tRNAs (L-aa-tRNAs) with both a high degree of speed and accuracy has been the focus of decades of intense mechanistic and structural investigations. In contrast, the response of the ribosome to D-aa-tRNAs has not been as well characterized. Nonetheless, a comprehensive mechanistic understanding of how the translational machinery (TM) responds to D-aa-tRNAs is of interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids. Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes and can be found at high concentrations in cells, and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids. Consistent with this possibility, the D-aa-tRNA deacylase (DTD) enzyme is nearly universally conserved and functions to remove D-amino acids that have been misacylated onto tRNA. [Emphasis added.]

Blocking the DTD allows D- forms to sneak in, but at only 17 percent the efficiency of the L- forms. This implies that something else besides DTD blocks them. Experiments showed that inside the ribosome, a “robust translation arrest event” occurs:

Consistent with its ability to induce translation arrest, chemical protection experiments and molecular dynamics (MD) simulations demonstrate that the presence of a D-amino acid at the C terminus of the nascent polypeptide chain can induce conformational changes within the ribosome that stabilizes the peptidyl-transferase center (PTC) in an inactive conformation.

In other words, the D-amino acid gums up the works in the translation factory. Something watches for these intruders and says, “Stop! You’re under arrest!” It puts up a physical barrier (conformational change) that prevents the prohibited alien from creating a bad protein. This appears to be a fail-safe system that can overcome failures at the DTD step.

By tweaking the translation machinery in their experiments, the scientists were able to get a ribosome to incorporate D-amino acids (phenylalanine, lysine, and valine) onto a polypeptide chain, “though the rate of dipeptide formation is orders of magnitude slower with the D-aa-tRNAs compared with their L-aa-tRNA counterparts.” This implies that the rejection of D-amino acids is due to mechanical constraints, not any mystical or vital force.

The methods used here establish an experimental framework for characterizing the precise mechanisms through which unnatural amino acids interfere with the function of the TM. The use of a purified in vitro translation system guaranteed the absence of DTD. Precautions were taken to ensure that tRNAs used to deliver un-natural amino acids to the TM were fully deacylated and devoid of their corresponding natural amino acids before aminoacylation with unnatural amino acids (Fig. S2 A).

What’s news is that the ribosome itself participates in the rejection of D-amino acids. If biochemists knock out the two safeguards, they can create novel polypeptides at will:

Collectively, these methods have allowed us to conclusively demonstrate that the ribosome itself discriminates the chirality of the amino acid and that, during continuous translation, the presence of a D-amino acid at the C terminus of a P site-bound peptidyl-D-aa-tRNA can arrest translation in a sub-population of elongating ribosomes. Notably, these methods have also enabled us to provide strong evidence indicating that the subpopulation of elongating ribosomes that are not translationally arrested by the presence of a P site-bound peptidyl-D- aa-tRNA should be able to continue translating unperturbed, resulting in the successful incorporation of D-amino acids into full-length proteins.

So they got the machines to sneak D-amino acids in. What of it? Is there a larger significance to this re-engineering test? They didn’t get into that. They were more focused on guiding “future efforts to engineer the TM so as to increase the efficiency with which D-amino acids, and possibly other unnatural amino acids, are incorporated into full-length proteins.” Notice that word “unnatural.” More on that later.

Certainly, this is valuable research. Beyond just playing around with genetic engineering, knowing how to re-engineer the TM to incorporate unnatural amino acids can help to understand and fight disease.

Given that D-amino acid racemase enzymes are found in the brain, that D-amino acids can be aminoacylated onto tRNA by aaRS, and that tRNA misacylation has been linked to neurodegenerative disorders, the translation disorders that we report here provide a plausible molecular mechanism that warrants future consideration as a potential underlying cause for neurodegenerative disease.

But as these tinkerers look into the mechanism to figure out how to hack it, we can step back and see a well-designed mechanical system oriented to produce 100 percent pure one-handed amino acids. We see at least two checkpoints: the DTD and the ribosome, whose job it is to maintain homochirality in a racemic chaos. Echoes of “Maxwell’s Demon” come to mind. This is not something that would happen naturally, unless a selecting agent were in operation to keep it that way. As in the Maxwell case, it doesn’t have to be a living agent; it can be a robotic system able to separate out molecules against the laws of thermodynamics. That requires intelligence, else it would be “unnatural.”

This finding also bears on the question of life’s origin. The authors didn’t discuss that, but it’s very important. Natural selection could not have operated before the first accurately replicating system arose. But without this complex proofreading and error-correcting equipment (made up of proteins and RNA) already in operation, functional proteins would be impossible. So how did the first proteins (and nucleic acids, which are also homochiral) arise from a primordial soup of racemic ingredients? The short answer is, they didn’t. The improbability of that occurring exceeds the Universal Probability Bound. An inference to intelligent causation is thus warranted.

Image: � Balint Radu / Dollar Photo Club.