B

Creation cf isologous interfaces leads to asymmetry

Figure 9.11. Heterologous interactions between subunits in a trimer.

part cf the ligand must interact with the active site cf the protein and "trigger" the interaction between subunits. In a heterotropic interaction parts of the regulatory ligand must provide direct binding energy for the binding and one part must again act as a trigger for the induced conformational change. In either case dissecting which parts of the ligand contribute to which types of interaction is critical in the design of an allosteric drug.

Over the last decade, major improvements in computer and graphics technology have facilitated the generation of numerous compu tational chemistry programs that allow investigators not only to view protein and small molecule structures, but also to use semiem-pirical parameters to quantitate atomic-level interactions between species of biomolecules (protein-protein, protein-ligand, protein-li-gand-water). One such program, HINT (Hydropathic INTeractions; eduSoft, LC, Richmond, VA), allows for the analysis of all possible noncovalent biomolecular atom-atom interactions, including hydrogen bonding, coulombic, acid-base, and hydrophobic forces. In brief, HINT employs thermodynamic,

Heterologous Isologous Interaction
Figure 9.12. Subunit contacts in aspartate transcarbamoylase show heterologous contacts in the catalytic trimers, isologous contacts between regulatory dimers, and regulatory subunit-catalytic subunit interactions. See color insert.

atom-based hydropathy values derived from solvent partitioning measurements of organic molecules, to quantitatively "score" interactions between the atoms of two species. These interactions can then be examined as color-coded contours.

If the crystal structure of the protein-li-gand complex is available, HINT analysis can yield valuable insight into specific contributions to the interaction. For example, in the binding of cofactor to the allosteric protein 3-phosphoglycerate dehydrogenase, HINT analysis, as shown in Fig. 9.13 indicates regions of both the protein surface and the co-

factor, which make either strong positive c negative contributions to the overall intera< tion. Use of this type of analysis as a startin point can lead to the design of analogs of th cofactor, or mutants of the protein, to furthe dissect contributions to binding and induce conformational changes. An understanding c the nature of ligand binding to an allosteri protein at this level is critical to the successfi design of an "allosteric" drug (see next se< Of equal importance is understandin the overall mechanism of transmission of th allosteric effect through the protein conformé tion to an adjacent subunit. As with the ana

Figure 9.13. HINT analysis of interactions between the cofactor NAD nicotinamide moiety and surrounding 3-phosphoglycerate dehydrogenase residues. Protein carbons are light gray and NAD carbons are black. Nitrogen atoms are blue and oxygen atoms are red. HINT contours are color coded :o represent the different types of noncovalent interactions as follows: blue = favorable hydrogen bonding and acid-base interactions; green = favorable hydrophobic-hydrophobic interactions; and -ed = unfavorable acid-acid and base-base interactions. The volume of the HINT contour represents he magnitude of the interactions. See color insert.

Figure 9.13. HINT analysis of interactions between the cofactor NAD nicotinamide moiety and surrounding 3-phosphoglycerate dehydrogenase residues. Protein carbons are light gray and NAD carbons are black. Nitrogen atoms are blue and oxygen atoms are red. HINT contours are color coded :o represent the different types of noncovalent interactions as follows: blue = favorable hydrogen bonding and acid-base interactions; green = favorable hydrophobic-hydrophobic interactions; and -ed = unfavorable acid-acid and base-base interactions. The volume of the HINT contour represents he magnitude of the interactions. See color insert.

ysis of ligand interactions with a protein, discussed above, a combination of computational and experimental approaches will be necessary to fully understand the mechanism of the transmission of an allosteric effect through a protein.

HINT analysis, in addition to being useful for the analysis of ligand-protein interactions, can lbe used to examine potential contributions to subunit interfaces in allosteric proteins. An example of such an analysis is shown in Fig. 9.14, where the cofactor binding domain subunit interface has been subjected to HINT analysis. Different regions of the sub-unite contribute either positively or negatively to the overall strength of the interface. Although in Hb, as discussed earlier, much detail is known about how the allosteric changes are ttriggered, this is not true for most alloste ric proteins. A few have had their three-dimensional structures determined in both the so-called R and T states, but even this has failed to give significant insight into the nature of the trigger or transmission of allosteric changes. As discussed above, computational analysis of crystal structures of oligomeric proteins and of ligand-protein complexes can give insight that can lead to tests by direct experimentation. Similar approaches are being developed to examine the ability of ligands to induce conformational changes. These involve docking substrates with the binding sites of the nonliganded form and using dynamics calculations to assess how the protein may readjust its conformation. The use of snapshots of these dynamic simulations are suggestive evidence of how the protein adjusts its conformation. If such approaches can be

Figure 9.14. HINT analysis of interactions between residues at the interface between two subunits of 3-phosphoglycerate dehydrogenase. The carbons of the two distinct subunits are colored light and dark gray, respectively. Nitrogen atoms are blue, oxygen atoms are red, and sulfur atoms are yellow. HINT contour maps visually displaying interactions between residues at the subunit interface are color coded according to the type of interaction: blue represents hydrogen bonding and favorable acid-base interactions, green displays favorable hydrophobic-hydrophobic contacts, red indicate; unfavorable base-base and acid-acid interactions, and purple indicates unfavorable hydrophobic-polar interactions. The volume of the HINT contour represents the magnitude of the interaction. For the analyzed phosphoglycerate dehydrogenase subunits, HINT indicates that a balance between favorable and unfavorable interactions characterizes the interface. See color insert.

Figure 9.14. HINT analysis of interactions between residues at the interface between two subunits of 3-phosphoglycerate dehydrogenase. The carbons of the two distinct subunits are colored light and dark gray, respectively. Nitrogen atoms are blue, oxygen atoms are red, and sulfur atoms are yellow. HINT contour maps visually displaying interactions between residues at the subunit interface are color coded according to the type of interaction: blue represents hydrogen bonding and favorable acid-base interactions, green displays favorable hydrophobic-hydrophobic contacts, red indicate; unfavorable base-base and acid-acid interactions, and purple indicates unfavorable hydrophobic-polar interactions. The volume of the HINT contour represents the magnitude of the interaction. For the analyzed phosphoglycerate dehydrogenase subunits, HINT indicates that a balance between favorable and unfavorable interactions characterizes the interface. See color insert.

validated by direct experimentation (e.g., using site-directed mutagenesis to change seemingly important residues in a predictable way), new insights into the triggers and transmission of allosteric changes will be obtained. With detailed information about both the triggers and transmission of allosteric conformational changes, the rational design of allosteric drugs will become more feasible.

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