Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition

Abstract

Several alanine mutations in the response regulator Spo0F induce hypersporulation in Bacillus subtilis. L66A, I90A and H101A mutants are purported to be involved in contacts stabilizing the orientation of the alpha4-helix and hence the beta4-alpha4 kinase recognition loop. Y13A is thought to affect the orientation of the alpha1-helix and consequently phosphatase action. Using comparative NMR chemical shift analyses for these mutants, we have confirmed these suppositions and isolated residues in Spo0F critical in sensor kinases discrimination. In addition, we discuss how buried residues and intra-protein communication networks contribute to precise molecular recognition by ensuring that the correct surface is presented.

Figure 1. Circular dichroism and tyrosine emission fluorescence

A) Far-UV circular dichroism spectra; B) tyrosine emission fluorescence of Spo0F mutants; “x” Y13A, “^” L66A, “*” I90A, “0” H101A and “+” wild type. Results confirm similar secondary structures between all mutant proteins and wild type, indicating no major conformational changes upon mutation.

Figure 2. Chemical shift changes for Spo0F mutants compared to wild-type

A: L66A; B: I90A; C: H101A; D: Y13A. The secondary structural elements (rectangles for helices, arrows for strands) were determined using the CSI program and ¹³Cα, ¹³Cβ, and ¹³CO chemical shift data. Δδ^NH was determined using Eqn. [1].

Figure 3. Residues in Spo0F mutants involved in kinase recognition

Residues in the β4-α4 kinase recognition loop exhibiting significant chemical shift perturbations (> 0.4ppm) mapped onto the structure of wild-type Spo0F (1FSP) [7]. Residues colored blue are perturbed in both L66A and I90A. Residues colored orange are only perturbed in L66A. Residues colored red are only perturbed in H101A. Residues colored green are only perturbed in I90A.