The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.

We have developed a method for calculating the association energy of quaternary complexes starting from their atomic coordinates. The association energy is described as the sum of two solvation terms and an energy term to account for the loss of translational and rotational entropy. The calculated solvation energy, using atomic solvation parameters and the solvent accessible surface areas, has a correlation of 96% with experimentally determined values. We have applied this methodology to examine intermediates in viral assembly and to assess the contribution isomerization makes to the association energy of molecular complexes. In addition, we have shown that the calculated association can be used as a predictive tool for analyzing modeled molecular complexes.Keywords: hydrophobicity; protein structure; solvation Specific interactions between macromolecules are responsible for the assembly of complex biological structures and are essential to the regulation of events within a cell or organism. The association of molecules to form higher ordered oligomers is in many respects analogous to the block condensation model for protein folding where prefolded units associate to form higher order structures (Richmond & Richards, 1978). To understand the structural basis of recognition we must be able to relate solution measurements of the association process to the structure of the macromolecular complex. This paper considers the problem of calculating the free energies of forming protein complexes from preformed subunits as derived from crystallographic data and relating these values to experimentally obtained association constants. Kauzmann (1959) suggested that a major factor in the stable formation of protein complexes is a consequence of the hydrophobic effect. Using an empirical correlation between the accessible surface area and free energies of transfer of amino acids from water to octanol, Chothia and Janin (1975) found that the free energy required to form a stable complex was directly related to the amount of surface area buried in the interface. Eisenberg and McLachlan (1986) recognized that it is an oversimplificaReprint requests to: Mitchell Lewis, The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104. tion to base the energy of association on surface area alone; polarity and charge must also be considered. By introducing five atomic solvation parameters, to account for the polar or apolar character of each atom type most frequently found in proteins, they could more accurately relate surface area to the free energy of transfer. Moreover, the solvation energy was shown to be useful for assessing protein stability. We assume that the forces that govern the association between two molecules are the same as the forces that are responsible for the folding of a protein in water. As such, the solvation energy should be equally useful as a gauge for evaluating the association energies of quaternary structu...