trp Repressor, A Crystallographic Study of Allostery in Genetic Regulation
Abstract of the lecture
The crystal structure of the E. coli trp repressor has been solved (1) and refined to 2.2 A. The two subunits (107 residues each) are related by an exact crystallographic dyad. Each subunit is composed of six helices, five of which intertwine about each other in a way that may make it seemingly impossible to disengage the subunits without altering their tertiary structure. The two symmetrically related L-tryptophan binding sites are formed by this interface.
Tryptophan must bind to the protein for repressor function. Tryptophan acts as an allosteric inhibitor as follows: (i) L-Tryptophan is wedged between the amino end of the C helix of one subunit and the side of the E helix of the dimer-related subunit fixing the orientation of the E helix, the most important element in recognizing the operator. (ii) The polar substituents of the bound tryptophan mold the protein’s polar residues near the region of the repressor surface where the DNA backbone most closely approaches the protein. (iii) The amino group of the tryptophan mitigates the negative charge potential arising from the carboxyl terminus of the B helix.
The crystal structure of trp aporepressor (2,3) has also been solved (by molecular replacement) and partially refined to 2.1 A. The aporepressor is the unliganded inactive form of the trp repressor that is activated to the repressor upon binding two molecules of L-tryptophan per dimer. By contrasting the aporepressor and repressor structures we can now define the allosteric transition that activates this sequence-specific DNA-binding regulatory protein.
The presence or absence of bound tryptophan has essentially no effect on the overall architecture, especially the dimer interface. However, if tryptophan is not bound, the helix-turn-helix motif ‘collapses’ into the ‘empty’ binding pocket. The largest differences between the structure of the liganded active repressor and that of the unliganded inactive aporepressor occur in the D-helix (the first helix of the motif), the amino terminus of the E helix (the second helix of the motif) and the intervening turn, that is, the residues involved in the repressor/operator interaction. There is also a substantial change in the conformation of the amino-terminal segment of the molecule.
Docking studies using a canonical B-DNA for the operator’s conformation show that the amino-terminal three residues of the E-helix, Ile79, Ala80 and Thr81, protrude into the major groove of the operator and make snug hydrophobic fit with the 5–6 region of the pyrimidines of the base pairs that are functionally most sensitive to mutational change. Electrostatic calculations (in collaboration with J. Warwicker, Yale) show a nearly perfect complementarity of charge potential in the complex. We report progress on the structure analysis of trp pseudorepressor (2,3) the adduct formed when certain analogues of L-tryptophan (e.g. indolepropionic acid) displace L-tryptophan. Although nearly isomorphous, the pseudorepressor does not bind selectively to the operator. Similarly we report progess on the structure analysis of the crystalline trp repressor/operator system.
KeywordsAllosteric Transition Dime Interface Major Groove Molecular Replacement Mutational Change