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:
Nucleosome: A Molecular Librarian, a Paradox
July, 2000 -- This is an auspicious time for molecular biology. The wave of knowledge that began in 1944 with Avery's discovery of DNA as the genetic material, which lead naturally to the atomic model of DNA proposed by Watson and Crick, and continued through detailed experiments to determine the genetic code, is now cresting with the release of the first draft of the human genome. This molecular text, written through billions of years of evolution, will provide untold insights into the molecular processes that underlie every aspect of our lives.
Each of our cells (or more correctly, nearly all of our cells) contain a copy of this genome, encoded in three billion base pairs of DNA. This information is precious and must be carefully guarded. Inside our cells, a collection of repair enzymes correct chemical changes inflicted on the strands by environmental insults. But the delicate strands must also be protected from physical damage. This is the job of nucleosomes.
The job of the nucleosome is paradoxical, requiring it to perform two opposite functions simultaneously. On one hand, nucleosomes must be stable, forming tight, sheltering structures that compact the DNA and keep it from harm. On the other hand, nucleosomes must be labile enough to allow the information in the DNA to be used. Polymerases must be allowed access to the DNA, both to transcribe messenger RNA for building new proteins and to replicate the DNA when the cell divides. The method by which nucleosomes solve these opposed needs is not well understood, but may involve a partial unfolding of the DNA from around the nucleosome, one loop at a time, as the information in the DNA is read.
Apart from their function of safely packaging DNA, nucleosomes also modify the activity of the genes that they store. Each nucleosome is composed of eight "histone" proteins bundled tightly together at the center, encircled by two loops of DNA. The histone proteins, however, are not completely globular like most other proteins. They have long tails, which comprise nearly a quarter of their length. The tails extend outward from the compact nucleosome, reaching out to neighboring nucleosomes and binding them tightly together. The nucleus contains regulatory enzymes that chemically modify these tails to weaken their interactions. In this way, the cell makes particular genes more accessible to polymerases, allowing their particular information to be copied and used to build new proteins.
The histone proteins are perfectly designed for their jobs, so much so that histones are nearly identical in all non-bacterial organisms. Even slight modifications can be lethal. The surface of the histone octamer is decorated with positively charged amino acids. These interact strongly with the negatively-charged phosphate groups on the DNA. This serves to glue the DNA strand to the protein core. This is no simple task. DNA is normally a long, straight molecule, but in nucleosomes the DNA must be forcibly bent into these two tight circles.
An intact nucleosome may be viewed in the PDB entry 1AOI. Keep in mind that this structure only includes a short piece of DNA. In reality, these little nucleosomes are arrayed by the millions along long strands of DNA.
Restriction Enzymes: Bacteria Fight Back with Molecular Scissors
August, 2000 -- Bacteria are under constant attack by bacteriophages, like the bacteriophage phiX174 described in an earlier Molecule of the Month. To protect themselves, many types of bacteria have developed a method to chop up any foreign DNA, such as that of an attacking phage. These bacteria build an endonuclease--an enzyme that cuts DNA--which is allowed to circulate in the bacterial cytoplasm, waiting for phage DNA. The endonucleases are termed "restriction enzymes" because they restrict the infection of bacteriophages.
Each type of restriction enzyme seeks out a single DNA sequence and precisely cuts it in one place. For instance, the enzyme EcoRI cuts the sequence GAATTC between the G and the A. Of course, roving endonucleases can be dangerous, so bacteria protect their own DNA by modifying it with methyl groups. These groups are added to adenine or cytosine bases (depending on the particular type of bacteria) in the major groove. The methyl groups block the binding of restriction enzymes, but they do not block the normal reading and replication of the genomic information stored in the DNA. DNA from an attacking bacteriophage will not have these protective methyl groups and will be destroyed. Each particular type of bacteria has a restriction enzyme (or several different ones) that cuts a specific DNA sequence, paired with a methyltransferase enzyme that protects this same sequence in the bacterial genome.
The booming field of biotechnology was made possible by the discovery of restriction enzymes in the early 1950's. With them, DNA may be cut in precise locations. A second enzyme--DNA ligase--may then be used to reassemble the pieces in any desired order. Together, these two enzymes allow researchers to assemble customized genomes. For instance, researchers can create designer bacteria that make insulin or growth hormone or add genes for disease resistance to agricultural plants.
An interesting property of restriction enzymes simplifies this molecular cutting and pasting. Restriction enzymes typically recognize a symmetrical sequence of DNA in which the top strand is the same as the bottom strand, read backwards. When the enzyme cuts the strand, it leaves overhanging chains termed "sticky ends" because the base pairs formed between the two overhanging portions will glue the two pieces together even though the backbone is cut. Sticky ends are an essential part of genetic engineering, allowing researchers to cut out little pieces of DNA and place them in specific places, where the sticky ends match.
The PDB contains structures for many restriction enzymes. The PDB entries 1RVC and 1RVA are two such examples of this type of molecule.
Lysozyme: A Cellular Guardian Attacking Bacteria
September, 2000 -- Lysozyme protects us from the ever-present danger of bacterial infection. It is a small enzyme that attacks the protective cell walls of bacteria. Bacteria build a tough skin of carbohydrate chains, interlocked by short peptide strands, that braces their delicate membrane against the cell's high osmotic pressure. Lysozyme breaks these carbohydrate chains, destroying the structural integrity of the cell wall. The bacteria burst under their own internal pressure.
Alexander Fleming discovered lysozyme during a deliberate search for medical antibiotics. Over a period of years, he added everything that he could think of to bacterial cultures, looking for anything that would slow their growth. He discovered lysozyme by chance. One day, when he had a cold, he added a drop of mucus to the culture and, much to his surprise, it killed the bacteria. He had discovered one of our own natural defenses against infection. Unfortunately, lysozyme is a large molecule that is not particularly useful as a drug. It can be applied topically, but cannot rid the entire body of disease, because it is too large to travel between cells. Fortunately, Fleming continued his search, finding a true antibiotic drug five years later: penicillin.
Lysozyme protects many places that are rich in potential food for bacterial growth. The lysozyme in the PDB entry 2LYZ is from hen egg whites, where it serves to protect the proteins and fats that will nourish the developing chick. It was the first enzyme ever to have its structure solved. Our tears and mucus contain lysozyme to resist infection of our exposed surfaces. Our blood is the worst place to have bacteria grow, as they would be delivered to all corners of the body. In the blood, lysozyme provides some protection, along with the more powerful methods employed by the immune system.
Lysozyme is a small, stable enzyme, making ideal for research into protein structure and function. Brian Matthews at the University of Oregon has performed a remarkable series of experiments, using lysozyme as the laboratory for study. He has performed hundreds of mutations on the lysozyme molecule made by a bacteriophage, changing one or more amino acids in the protein chain to a different one. He has studied the effect of removing large residues inside the protein, leaving a hole, or cramming a large amino acid inside, where it would not normally fit. He has attempted to create new active sites by creating new molecule-shaped pockets. Structures of hundreds of these mutant lysozymes are available at the PDB- -so many, in fact, that lysozyme is the most common protein in the PDB. The structure from PDB entry 1L35 is a mutant. Two of its amino acids have been changed to cysteine, forming a new disulfide bridge in the mutation. The native enzyme can be found in the PDB entry 1LYD.
Lysozyme has a long active site cleft that binds to the bacterial carbohydrate chain. Based on computer modeling, it has been proposed that lysozyme distorts the shape of one sugar ring in the chain, making it more easy to cleave (although other studies have proposed that different effects, like electrostatics, are more important). The structure found in PDB entry 148L shows what this distorted ring might look like. Normally, sugar rings adopt a zig-zag "chair" structure.
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