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.

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...

SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma & Bitinaite, (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR|CCGGYG, | indicates cut site, R=A,G, Y=C,T) and secondary (CR|CCGGY(A/C/T) and CR|CCGGGG) site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, et al., (2008) Nucleic Acids Res. 36, 5405–5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers (High Molecular Weight Species, HMWS) in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence, that are dependent on the concentration of the DNA bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15–20 s, and is composed of possibly 4–12 DNA bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself, but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA bound SgrAI (i.e. HMWS) are the activated, or activatable, form of the enzyme.

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca 2؉ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an innersphere water molecule to the phosphate group located 3 to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-S P phosphoryl oxygen on the adjacent 3 phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.Recently determined crystal structures of type II restriction endonucleases have produced a wealth of information on the basis for target site sequence selectivity (1-6). However, these structures do not resolve the detailed structural mechanism of catalysis. Although acidic and basic groups in the active sites can be identified, and in some cases divalent-metal binding sites delineated, a convincing picture clarifying the way in which the attacking hydroxide ion is generated, and the leaving group stabilized, has not been elucidated for any of the enzymes.EcoRV endonuclease is a homodimer of 244 aa per monomer. It cleaves the duplex sequence 5Ј-GATATC at the central TA step in a blunt-ended fashion (7). As in all type II enzymes, phosphoryl transfer proceeds via attack of a hydroxide ion nucleophile on the scissile phosphorus, generating products containing a 5Ј phosphate group. This reaction occurs by in-line displacement through a pentacovalent transition state, with inversion of stereochemistry at phosphorus and an absolute requirement for divalent metal cations (8, 9). Highresolution crystallographic analyses of EcoRV show that the scissile phosphates of the DNA are located adjacent to the carboxylates of Asp-90 and Asp-74, and that a divalent metal ion bridges the enzyme and DNA at this position (2, 3). Although this metal could stabilize the additional negative charge that develops in the transition state, it is not correctly positioned to generate the attacking hydroxide ion. Thus, speculative mechanisms in which the nucleophile arises from dissociation of a metal-ligated water have invoked significant rearrangements of the DNA from its observed conformation in the crystal structures (2, 10).An alternative mechanism involving substrate-assisted catalysis by the adjacent 3Ј-phosphate of the DNA also has been proposed (11,12). In support of this mechanis...