Long range dynamic effects of point-mutations trap a response regulator in an active conformation

Abstract

When a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This 'conformational trapping' results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site.

Fig. 1

NMR structures of L66A, I90A and H101A. Ensembles of the 10 lowest energy structures of L66A (A), I90A (B), and H101A (C) shown through stereo images rendered through PyMOL (http://www.pymol.org).

Fig. 2

(A) overlay of inactive wt Spo0F (two structural families - blue and green) with active wt Spo0F-BeF₃ (pink); (B) expansion of the β4-α4 loop (same color scheme); (C) same as (A) but now including H101A Spo0F strcuture (orange) - representative of hypersporulation mutants; (D) expansion of the β4-α4 loop (same color scheme).

Fig. 3

(A) Conformational transitions simulation of inactive wt Spo0F. The two structures with the greatest RMSD over the simulation are shown in green and red. Black arrows highlight the magnitude of this difference in the β4-α4 loop. (B) Conformational transitions simulation of constitutively active L66A Spo0F - same color scheme as (A). (C) Principal components for residues in kinase recognition cluster 6. Symbols are as follows: blue “” Spo0F; red “” Spo0F-BeF₃; orange “” L66A; purple “” I90A; black “*” H101A.