|
The Giovanni Armenise-Harvard Foundation Session 5: Structural Biology and Enzymology Overview Since the 1930s, scientists have used X-ray crystallography to visualize the 3-D structure of enzymes and other proteins at the atomic level. Although this technique has been refined considerably over six decades, the basic idea has remained constant: when a narrow beam of X-rays is focused on a pure crystal of protein, the atoms will scatter the waves in the beam and create an X-ray diffraction pattern that reveals the relative position of atoms are in the molecule. A second approach, nuclear magnetic resonance (NMR) spectroscopy, has been used to study protein structure since the 1980s. Although it has the advantage of not requiring pure protein crystals (which are famously difficult to prepare), it can only be used to analyze very small proteins. The presentations in this section of the symposium concern the three-dimensional structures of two enzymes important for cell growth and division, a transcription factor, enzymes essential for DNA repair, and a viral toxin that is important in the pathogenesis of stomach ulcers.
Normal Src is a receptor tyrosine kinase that receives extracellular growth signals and turns them into signals that can be read by intracellular messengers. SRC accomplishes this by phosphorylation, which involves tacking phosphate groups onto tyrosine amino acids in other proteins. Although Src normally switches on and off as needed, research has shown that oncogenic mutations can lock Src into the "on" position- which sends the cell into a growth-promoting frenzy. The exact mechanism for this is unclear, although researchers suspect that Src is a multiplex switch capable of turning on or off in response to diverse types of input. Dr. Eck's laboratory has produced a 3-D structure of the oncogene Src that is the highest resolution image ever made of a protein of this class. Of the protein's four distinct lobes, two make up the kinase that phosphorylates other proteins and two others, dubbed SH2 and SH3, regulate the kinase and help Src establish its site of action within the cell. The researchers found that several mechanisms work simultaneously to keep Src idle, but once it is activated its four lobes curl tightly around the active site-making it inaccessible to an "off" signal. This information could prove useful to drug designers in the long run.
In another study of the relationship between structure and function, Dr. Pinna's group examined the atomic details of a protein kinase called CK2. This is an essential, ubiquitous, and pleiotropic enzyme that is known to act on more than 150 substrates. Most interesting to cancer researchers is that its overexpression correlates with neoplastic growth. Also CK2 at first appears of be a single structure, in fact it is formed by the tight and stable association of two catalytic ( a and/or aÎ) and two modulatory §-subunits. Close examination reveals that the structure of CK2? has features that give it properties that are unique among protein kinases: it binds both ATP and GTP, in their syn rather than anti conformation; it recognizes phosphoacceptor sites marked by multiple acidic residues; and it has high basal activity due to displacement of the "activation loop" which interacts in a stable fashion with the N-terminal segment. Additional studies, using mutants and synthetic fragments of the §-subunits, demonstrate that they have both positive and negative regulatory properties.
Transcription factors switch on genes, or groups of genes, so that they generate the messenger RNA that is needed to produce proteins that the cell needs for growth. Transcriptional control is a key step because it determines when and how often a given gene is transcribed. It appears that instead of always coming from a single source, transcriptional commands sometimes take the form of integrated signals that come from several pathways. This suggests that when genes are inappropriately transcribed, the fault may be a mutation in one transcription factor or a flawed interaction between two or more of these regulatory substances. Dr. Harrison and his team have studied how synthesis of the T-cell cell growth factor interleukin-2 (IL-2) is regulated, and have learned that control comes not from a single factor, but rather from a complex of proteins working together. Transcription of the IL-2 gene is predominantly regulated by nuclear factor of activated T-cells (NF-AT), which is activated in the cytoplasm of T-cells after a receptor has been stimulated by calcineurin. It forms a transcriptional complex with an activator protein,AP-1, which is a heterodimer of fos and jun oncoproteins. The researchers have determined the three-dimensional structure of a quaternary complex of NF-AT, fos, jun, and DNA. The interactions among these components are striking and extensive.
Chemical assailants constantly chip away at the integrity of DNA, increasing the chance that it will be transcribed into defective proteins that will harm the body. Cells respond to this threat by dispatching crews of repair enzymes, which mend damaged areas by cutting out bad parts, making a new copy of genetic information that was destroyed, and sealing gaps. Dr. Ellenberger and his colleagues have done 3-D structures that reveal some of the details of the repair process. X-ray structures of a base excision-repair enzyme (AlkA) and a DNA polymerase (T7 DNA polymerase) show how these enzymes go about repairing DNA. DNA repair is almost always desirable, because without it harmful mutations would accumulate and pose a threat to life itself. The exception comes in cancer cells which are under therapeutic attack by chemotherapy agents, In this case, the ability of tumor cells to repair damaged DNA often limits the effectiveness of therapeutic agents. Knowing more about the 3-D structure of repair enzymes could prove especially relevant to cancer research.
Heliobacter pylori (HP) is a corkscrew-shaped bacterium that lives in the gastrointestinal tract of 50% of the population, but causes symptomatic illness in only 10%. For that unfortunate minority, it can cause a wide range of troubles including gastritis, stomach and duodenal ulcer, and sometimes even adenocarcinoma or mucosal-associated lymphoid tissue cancers. The fact that not everyone who is infected becomes ill raises the possibility that some HP strains may have virulence factors that make them more dangerous than others. There is high degree of interest in creating vaccines that could block HP infection or mediate its effects, and in designing better treatments for the common diseases the bacterium causes. Rational design of such agents could be advanced by more detailed information about virulence factors and their impact on the cells of the human gut. HP produces three main virulence factors: Aurease, essential for its survival in the stomach; VacA, which induces intracellular vacuolation that causes host cells to collapse and die; and CaqA, about which less is known. VacA is a toxin that is associated with more severe symptoms. In an effort to clarify its action, Dr. Papini added purified VacA to established cell lines and observed the morphological and functional alterations it induced. Although many bacterial change the cytosol of target cells, VacA appears to disrupt the structure and function of the cell's endocytic pathway in unique ways.
|
Structure and regulation of human C-SRC tyrosine kinase
