Environment‐dependent long‐range structural distortion in a temperature‐sensitive point mutant

Abstract

Extensive environment-dependent rearrangement of the helix-turn-helix DNA recognition region and adjacent L-tryptophan binding pocket is reported in the crystal structure of dimeric E. coli trp aporepressor with point mutation Leu75Phe. In one of two subunits, the eight residues immediately C-terminal to the mutation are shifted forward in helical register by three positions, and the five following residues form an extrahelical loop accommodating the register shift. In contrast, the second subunit has wildtype-like conformation, as do both subunits in an isomorphous wildtype control structure. Treated together as an ensemble pair, the distorted and wildtype-like conformations of the mutant apoprotein agree more fully than either conformation alone with previously reported NOE measurements, and account more completely for its diverse biochemical and biophysical properties. The register-shifted segment Ile79-Ala80-Thr81-Ile82-Thr83 is helical in both conformations despite low helical propensity, suggesting an important structural role for the steric constraints imposed by b-branched residues in helical conformation.

Multiple helical conformations of the helix‐turn‐helix region revealed by NOE‐restrained MD simulations of tryptophan aporepressor, TrpR

“…The fact that the measured value is approximately 2.6‐fold lower than predicted thus suggests that part of the population is not in a binding‐compatible conformation. The distorted conformer of WT apoTrpR is predicted to be incompatible with L‐trp binding because Ile82 occludes the L‐trp binding site of the distorted monomer in the L75F apoTrpR crystal structure . Hence, the presence of this and/or other binding‐incompatible conformations is consistent with the slower than expected on‐rate of L‐trp binding.…”

“…The starting structure of WT apoTrpR in WT conformation was prepared from the 1.1 Å crystal structure PDB ID 3SSW, the highest resolution crystal structure of apoTrpR available. Using symmetry operations in YASARA the subunits of the crystal were made to have identical conformations.…”

“…A crystal structure of temperature‐sensitive TrpR mutant apoL75F (PDB ID: 3SSX) revealed a distorted conformation in the HtH region on one subunit of the dimer (Fig. B).…”

“…B). The other subunit is in WT conformation, as are both subunits in isomorphous WT apoTrpR crystals . In the distorted conformation, helix D undergoes a rigid‐body shift of 3 Å relative to the WT conformation; residues 76–79 in the D‐E turn form a 3 10 helix; and helix E has a local interruption with residues 85–88 looping out.…”

“…The finding that the distorted conformation is not likely to be an artefact of crystallization suggests that this conformation might be present in solution. To evaluate this possibility, NOE data for apoL75F were re‐analyzed, uncovering evidence for the presence in solution of both the conformations observed in the L75F crystal structure. That finding highlighted a major limitation of current NMR structure determination methods, which neglect conformational dynamics by requiring every structure in the ensemble to be consistent with all NOEs.…”