 |
Structural Basis of Interactions Within and Between Macromolecules
Summary: Brian Matthews uses x-ray crystallography and other techniques to study the mechanisms of protein folding and protein-protein, protein-ligand, and DNA-protein interactions.
Our laboratory uses x-ray crystallography, in concert with other techniques, to address fundamental problems in biology: How do proteins spontaneously fold into their biologically active three-dimensional configurations? What determines the stability of these folded proteins? Can stability be improved? How do proteins interact with each other? How do proteins interact with DNA? How do enzymes act as catalysts? Significant progress has been made in simplifying the protein-folding "problem," in quantitating the interactions that stabilize protein structures, and in enhancing our understanding of protein-protein, protein-ligand, and DNA-protein interactions.
Protein Folding, Stability, and Design
We believe that the T4 lysozyme system will continue to be very useful to address a variety of questions related to protein folding, stability, and function. Our ultimate objective is to understand how the amino acid sequence of a protein determines its three-dimensional structure.
We are continuing to simplify the protein-folding problem by identifying which residues, or combinations of residues, are critical for the folding and stability of T4 lysozyme. We want to understand not only how given residues contribute to stability but also the relative importance of local versus nonlocal effects in protein folding. In other words, we want to determine the degree to which the structures of local segments of the polypeptide chain are determined by the amino acid sequence of that segment, and to what extent by the structural context provided by the rest of the folded protein.
In a set of experiments, in collaboration with Brian Shoichet's group (University of California, San Francisco), we are using engineered cavities in T4 lysozyme to test and develop docking procedures. The objective is to take a large library of known small-molecule compounds and to analyze these computationally to determine which might bind to a known receptor site. To test these docking procedures more rigorously, we have constructed not only nonpolar cavities but also variants that have been engineered to include polar functionalities. Appropriate consideration of protein and ligand desolvation has been found to help discriminate between putative ligands. We are now focusing on the need to allow for structural changes in the protein that may accompany ligand binding.
Are Nonpolar Cavities in Proteins Empty?
The extent to which water is present within apolar cavities in proteins remains unclear. In the case of interleukin-1β, four independent structures solved by x-ray crystallography agree that water is not present in the central apolar cavity. In contrast, results from NMR (nuclear magnetic resonance) spectroscopy suggest that water has high occupancy within the cavity but is positionally disordered, making it undetectable by standard crystallographic methods. To resolve these discrepancies we sought to obtain an experimentally phased electron density map that was free of possible bias due to mathematical modeling of the protein or the solvent. By combining native diffraction data with multiple wavelength anomalous data from a platinum derivative, we obtained accurate phases. Using these experimental phases, we estimate that occupancy of the apolar cavity in interleukin-1β by solvent is close or equal to zero. Polar cavities in the protein that contain ordered solvent molecules serve as internal controls.
The mutant Leu99 → Ala (L99A) in T4 lysozyme results in a nonpolar cavity of volume about 150 Å3. There has been ongoing controversy as to whether this cavity is really empty or contains disordered solvent (water) molecules. In collaboration with Sol Gruner and Marcus Collins (Cornell University) and Gerhard Hummer (National Institutes of Health), we have investigated the effect of pressure on the structure of the L99A mutant. At about 2,000 atm, four water molecules are seen to enter the cavity, while the protein itself remains essentially rigid. Molecular dynamics simulations show that the filling is highly cooperative and is primarily due to a 4 kJ/mol change in bulk water activity. These studies all tend to confirm that nonpolar cavities in proteins are empty unless they are sufficiently large to accommodate three or more water molecules that can hydrogen-bond to each other.
DNA-Protein Interaction
Our laboratory has maintained a long-standing interest in protein-DNA interactions. In collaboration with Chris Doe (HHMI, University of Oregon), we have also been attempting structural studies of proteins involved in asymmetric cell division. Three proteins—Miranda, Staufen, and Prospero—are key players in this process. Most progress has been made with structural studies of Prospero. Capitalizing on its predicted domain structure, we overexpressed the carboxyl-terminal 173 amino acids and determined the structure. This region of the protein was found, somewhat unexpectedly, to consist of a homeodomain plus the so-called Prospero domain combined in a single homeo-Prospero domain (HPD). Recently we have determined the structure of the HPD in complex with its DNA target. Knowledge of these structures will facilitate genetic analysis related to function.
Protease Structure and Function
We have been interested in the methionine aminopeptidases (MetAPs) because they are essential in some microorganisms and therefore might be potential targets for novel antibiotics. The human type II MetAP has also been shown to be the target of certain inhibitors of angiogenesis that have undergone clinical trials for a variety of cancers. We initially determined the structure of the prototypical MetAP from Escherichia coli, followed by other family members. Our determination of the structure of type I human MetAP made it possible, for the first time, to compare the structures of a type I and a type II MetAP from the same organism and to explain why ovalicin and related anti-angiogenesis inhibitors target type II human MetAP but not type I.
Protein degradation is an essential function in all living cells but needs to be carefully regulated. Proteins destined for degradation are tagged, unfolded, and broken down in a series of steps. Aminopeptidase N is a major metalloprotease that participates in this final step in E. coli. Determination of its 870–amino acid structure revealed a thermolysin-like active site enclosed within a large cavity of volume 2,200 Å3 that is inaccessible to substrates except for a small opening of about 8–10 Å. These results, combined with the results of others, indicate that proteases that are involved in intracellular peptide degradation prevent inadvertent hydrolysis of inappropriate substrates by enclosing the active site within a large cavity. There is also some evidence that the "open" form of the enzyme, which admits substrates, remains inactive until it adopts the closed form.
Some studies related to protein stability were supported in part by grants from the National Institutes of Health.
Last updated March 09, 2007
|
|
|
|
