Molecules of the Quarter:
Aminoacyl-tRNA Synthetases, Cyclooxygenase, and Myosin

The PDB has continued to feature its popular "Molecule of the Month" piece. Written and drawn by David S. Goodsell, an assistant professor of molecular biology at The Scripps Research Institute in La Jolla, California, these articles provide an overview of significant milestones in the growth of the PDB's macromolecular structure data for a diverse audience. Here is a sample of the information that is presented in this feature:

Aminoacyl-tRNA Synthetases: Twenty Flavors

PDB ID: 1asz

J. Cavarelli, G. Eriani, B. Rees, M. Ruff, M. Boeglin, A. Mitschler, F. Martin, J. Gangloff, J.C. Thierry, D. Moras (1994): The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction. EMBO. J. 13, p. 327.

April, 2001 -- When a ribosome pairs a "CGC" tRNA with "GCG" codon, it expects to find an alanine carried by the tRNA. It has no way of checking; each tRNA is matched with its amino acid long before it reaches the ribosome. The match is made by a collection of remarkable enzymes, the aminoacyl-tRNA synthetases. These enzymes charge each tRNA with the proper amino acid, thus allowing each tRNA to make the proper translation from the genetic code of DNA into the amino acid code of proteins.

Most cells make twenty different aminoacyl-tRNA synthetases, one for each type of amino acid. These twenty enzymes are widely different, each optimized for function with its own particular amino acid and the set of tRNA molecules appropriate to that amino acid. The one shown here, which charges aspartic acid onto the proper tRNA (PDB entry 1asz), is a dimer of two identical subunits (colored blue and green, the two tRNA molecules are colored red). Others are small monomers or large monomers, or dimers, or even tetramers of one or more different types of subunits. Some have wildly exotic shapes, such as the serine enzyme (PDB entry 1set). The structures of nearly all of these different enzymes are available in the PDB.

As you might expect, many of these enzymes recognize their tRNA molecules using the anticodon. But this may not be possible in some cases. Take serine, for instance. Six different codons specify serine, so seryl-tRNA synthetase must recognize six tRNA molecules with six different anticodons, including AGA and GCU, which are entirely different from one another. So, tRNA molecules are also recognized using segments on the acceptor end and bases elsewhere in the molecule. One base in particular, number 73 in the sequence, seems to play a pivotal role in many cases, and has been termed the discriminator base. In other cases, however, it is completely ignored. Note also that each enzyme must recognize its own tRNA molecules, but at the same time, it must not bind to any of the other ones. So, each tRNA has a set of positive interactions that match up the proper tRNA with the proper enzyme, and a set of negative interactions that block binding of improper pairs. For instance, the aspartyl-tRNA synthetase found in PDB entry 1asz recognizes the discriminator base and 4 bases around the anticodon. But, one other base, guanine 37, is not used in binding, but must be methylated to ensure that the tRNA does not bind improperly to the arginyl-tRNA synthetase.

Recent analyses of entire genomes revealed a big surprise: some organisms don't have genes for all twenty aminoacyl-tRNA synthetases. They do, however, use all twenty amino acids to construct their proteins. The solution to this paradox revealed, as is often the case in living cells, that more complex mechanisms are used. For instance, some bacteria do not have an enzyme for charging glutamine onto its tRNA. Instead, a single enzyme adds glutamic acid to all of the glutamic acid tRNA molecules and to all of the glutamine tRNA molecules. A second enzyme then converts the glutamic acid into glutamine on the latter tRNA molecules, forming the proper pair.

Aminoacyl-tRNA synthetase enzymes approach the tRNA from different angles. Isoleucine (entry 1ffy), valine (entry 1gax) and glutamine (entry 1euq) enzymes cradle the tRNA, gripping the anticodon loop (at the bottom in each tRNA), and placing the amino-acid acceptor end of the tRNA in the active site (at the top right in each tRNA). These all share a similar protein framework, known as "Type I," approaching the tRNA similarly and adding the amino acid to the last 2' hydroxyl group in the tRNA. The phenlyalanine (entry 1eiy) and threonine (entry 1qf6) enzymes are part of a second class of enzymes, known as "Type II." They approach the tRNA from the other side, and add the amino acid to the other free hydroxyl on the last tRNA base.

Aminoacyl-tRNA synthetases must perform their tasks with high accuracy. Every mistake they make will result in a misplaced amino acid when new proteins are constructed. These enzymes make about one mistake in 10,000. For most amino acids, this level of accuracy is not too difficult to achieve. Most of the amino acids are quite different from one another, and, as mentioned before, many parts of the different tRNA are used for accurate recognition. But in a few cases, it is difficult to choose just the right amino acids and these enzymes must resort to special techniques.

Isoleucine is a particularly difficult example. It is recognized by an isoleucine-shaped hole in the enzyme, which is too small to fit larger amino acids like methionine and phenylalanine, and too hydrophobic to bind anything with polar sidechains. But, the slightly smaller amino acid valine, different by only a single methyl group, also fits nicely into this pocket, binding instead of isoleucine in about 1 in 150 times. This is far too many errors, so corrective steps must be taken. Isoleucyl-tRNA synthetase (PDB entry 1ffy) solves this problem with a second active site, which performs an editing reaction. Isoleucine does not fit into this site, but errant valine does. The mistake is then cleaved away, leaving the tRNA ready for a properly-placed leucine amino acid. This proofreading step improves the overall error rate to about 1 in 3,000.

These enzymes are not gentle with tRNA molecules. The structure of glutaminyl-tRNA synthetase with its tRNA (entry 1gtr) is a good example. The enzyme firmly grips the anticodon, spreading the three bases widely apart for better recognition. At the other end, the enzyme unpairs one base at the beginning of the chain, and kinks the long acceptor end of the chain into a tight hairpin. This places the 2' hydroxyl on the last nucleotide in the active site, where ATP and the amino acid are bound.

A list of Aminoacyl-tRNA Synthetases in the PDB as of April, 2001, is available at For suggestions for further information on aminoacyl-tRNA synthetases, please see

Cyclooxygenase: A Complex Enzyme

PDB ID: 1prh

D. Picot, P.J. Loll, R.M. Garavito (1994): The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature 367, p. 369.

May, 2001 -- What is the most commonly-taken drug today? It is an effective painkiller. It reduces fever and inflammation when the body gets overzealous in its defenses against infection and damage. It slows blood clotting, reducing the chance of stroke and heart attack in susceptible individuals. And, there is growing evidence that it is an effective addition to the fight against cancer. This wonder drug, with manifold uses in medicine, is aspirin. Aspirin has been used professionally for a century, and traditionally since ancient times. A similar compound found in willow bark, salicylic acid, has a long history of use in herbal treatment. But only in the last few decades have we understood how aspirin works, and how it might be improved.

As you might expect from a drug with such diverse actions, aspirin blocks a central process in the body. Aspirin blocks the production of prostaglandins, key hormones that are used to carry local messages. Unlike most hormones, which are produced in specialized glands and then delivered throughout the body by the blood, prostaglandins are created by cells and then act only in the surrounding area before they are broken down. Prostaglandins control many of these neighborhood processes, including the constriction of muscle cells around blood vessels, aggregation of platelets during blood clotting, and constriction of the uterus during labor. Prostaglandins also deliver and strengthen pain signals and induce inflammation. These many different processes are all controlled by different prostaglandins, but all created from a common precursor molecule.

Cyclooxygenase (shown here from PDB entry 1prh) performs the first step in the creation of prostaglandins from a common fatty acid. It adds two oxygen molecules to arachidonic acid, beginning a set of reactions that will ultimately create a host of unusual molecules. Aspirin blocks the binding of arachidonic acid in the cyclooxygenase active site. The normal messages are not delivered, so we don't feel the pain and don't launch an inflammation response.

We actually build two different cyclooxygenases (termed COX-1 and COX-2) for different purposes. COX-1 is built in many different cells to create prostaglandins used for basic housekeeping messages throughout the body. The second enzyme is built only in special cells and is used for signaling pain and inflammation. Unfortunately, aspirin attacks both. Since COX-1 is targeted, aspirin can lead to unpleasant complications, such as stomach bleeding. Fortunately, specific compounds that block just COX-2, leaving COX-1 to perform its essential jobs, are now becoming available. These new drugs are selective pain-killers and fever reducers, without the unpleasant side-effects.

This enzyme actually has two different active sites, collectively termed prostaglandin synthase. On one side, it has the cyclooxygenase active site discussed previously. On the opposite side, is has an entirely separate peroxidase site, which is needed to activate the heme groups that participate in the cyclooxygenase reaction. The enzyme complex is a dimer of identical subunits, so altogether, there are two cyclooxygenase active sites and two peroxidase active sites in close proximity. Each subunit has a small carbon-rich knob. These knobs anchor the complex to the membrane of the endoplasmic reticulum. The cyclooxygenase active site is buried deep within the protein, and is reachable by a tunnel that opens out in the middle of the knob. This acts like a funnel, guiding arachidonic acid out of the membrane and into the enzyme for processing. In the structure found at PDB entry 4cox, a drug is blocking the active site in both subunits. The heme groups are also shown above the drug in each subunit.

PDB entry 1pth shows how aspirin blocks the cyclooxygenase active site. Aspirin is composed of two parts: an acetyl group attached to salicylic acid. When it attacks cyclooxygenase, it connects its acetyl group to a serine amino acid, permanently inactivating the enzyme. After aspirin has performed its job, the acetyl group becomes attached to the serine amino acid, and the salicylic acid is bound close by.

A list of all cyclooxygenases as of May, 2001, is available at For suggestions for further reading on cyclooxygenase, please see

Myosin: Molecular Motion
PDB ID: 1b7t

A. Houdusse, V.N. Kalabokis, D. Himmel, A.G. Szent-Gyorgyi, C. Cohen (1999): Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head. Cell 97, p. 459.




Each myosin performs only a tiny molecular motion. It takes about 2 trillion myosin molecules to provide the force to hold up a baseball. Our biceps have a million times this many, so only a fraction of the myosin molecules need to be exerting themselves at any given time. By working together, the tiny individual power stroke of each myosin is summed to provide macroscopic power in our familiar world. Inside muscle cells, about 300 myosin molecules bind together, with all of the long tails bound tightly together into a large "thick filament." The many myosin heads extending from the thick filament then reach over to actin filaments, and together climb their way up.

ATP contains a key phosphate-phosphate bond that is difficult to create and is used to power many processes inside cells. You might be surprised to find, however, that breakage of this phosphate-phosphate bond is not directly responsible for the power stroke in myosin. Instead, it is release of the phosphate left over after ATP is cleaved that powers the stroke. Think of myosin like an arm that can flex towards you or push away. The cleavage of ATP is used in a priming step. When ATP is cleaved, myosin adopts a bent, flexed form, like in PDB entry 1br1. This prepares myosin for the power stroke. The flexed myosin then grabs the actin filament and release of phosphate snaps it into the straight "rigor" form. This power stroke pushes the myosin molecule along the actin filament. When finished, the remaining ADP is replaced by a new ATP, the myosin lets go of the actin filament. Then, it is ready for the next stroke.

The myosin motor domain, from entry 1b7t, is nearly straight, close to the rigor form. You can explore several interesting features. At the tip of the molecule is a cleft that binds to the actin filament. Notice that the ADP molecule is bound at the base of this deep cleft. It is thought that changes in the nucleotide, as it cycles from ATP, to ADP and phosphate, to ADP alone, are transmitted along this cleft to change the way that myosin interacts with actin. In the middle of the molecule is the "converter" domain that changes shape when phosphate is released. On one side of the molecule is a long alpha helix with the two light chains bound around it. This is the "lever arm" that amplifies the converter shape change into a large power stroke.

A list of all myosin structures as of June, 2001, is available at For suggestions for further reading about myosin, please see