Dihydrofolate reductase protein interactions

The author assumed that the Born radii of atoms can be estimated from the solvent exposure factors for sampling spheres around the atoms. Two spheres were used in a five-parameter equahon to calculate the Born radii. The parameters of the equahon were eshmated using numerical calculahons from X-ray protein structures for dihydrofolate reductase. In addition to AGol the author also considered the AGJ term accounting for cavity formahon and dispersion of the solute-solvent interactions as ... 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

While providing valuable insight towards a better understanding of protein evolution, the bisection of proteins into functional heterodimers has also found practical applications to study protein-protein interactions. Heterodimeric variants of dihydrofolate reductase (DHFR), green fluorescent protein (GFP), GAR transformylase, and phosphotransferases were constructed to work in two-hybrid systems [64]. 

The Michnick group has also developed an elegant split-reporter system, based on reconstitution of dihydrofolate reductase (DHFR) (41). In this system, cell survival requires functional DHFR thus, a successful protein-protein interaction can be identified readily by selection. This system has been applied to the identification of peptide sequences that bind the Ras-binding domain of Raf (42). 

Remy 1, Campbell-Valois FX, Michnick SW. Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc. 2007 2 2120-2125. 

Covalent bonds are not as important in drug-receptor binding as noncovalent interactions. Alkylating agents in chemotherapy tend to react and form an immonium ion, which then alkylates proteins, preventing their normal participation in cell divisions. Baker s concept of active site directed irreversible inhibitors was well established by covalent formation of Baker s antifolate and dihydrofolate reductase (46). 

W discuss three examples of "drug target" interactions (l)biotin-avidin (2) dihydrofolate reductase-trimethoprim, and (3)DNA-in-tercalator. The first is the strongest characterized protein-ligand association, the second a prototype enzyme-inhibitor interaction, and the third describes drugs interacting with nucleic acids. 

An extensively studied enzyme-inhibitor system involves the protein dihydrofolate reductase/ and demonstrates an important feature of drug-enzyme interactions - the fact that an inhibitor drug may not... 

FIGURE 6-4 Complementary shapes of a substrate and its binding site on an enzyme. The enzyme dihydrofolate reductase with its substrate NADP" (red), unbound (top) and bound (bottom). Another bound substrate, tetrahydrofolate (yellow), is also visible (PDB ID 1 RA2).The NADP binds to a pocket that is complementary to it in shape and ionic properties. In reality, the complementarity between protein and ligand (in this case substrate) is rarely perfect, as we saw in Chapter 5. The interaction of a protein with a ligand often involves changes in the conformation of one or both molecules, a process called induced fit. This lack of perfect complementarity between enzyme and substrate (not evident in this figure) is important to enzymatic catalysis. 

Significant progress in QSAR resulted from Hansch analyses of enzyme inhibitors [432, 456, 668 — 670], especially from the systematic work of Hansch and his group on dihydrofolate reductase and on cysteine and serine proteases. Most of our current knowledge of the quantitative aspects of ligand-protein interactions has been derived from QSAR equations, aided by the interpretation of the 3D structures of enzymes and their inhibitor complexes with molecular graphics [38, 288, 671 — 676]. 

An extensively studied enzyme-inhibitor system involves the protein dihydrofolate reductase [88]. Crystallographic results demonstrate an important feature of drug-enzyme interactions an inhibitor drug may not bind in the same way as substrate even though both have similar chemical formulae. This enzyme catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential... 

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