Transmembrane Prolines Mediate Signal Sensing and Decoding in Bacillus subtilis DesK Histidine Kinase

Abstract

Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below ∼30°C. Membrane fluidity signals are transmitted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of membrane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased mutagenic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every transmembrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the metastable 2-HCC domain of the DesK signaling state.IMPORTANCE Signal sensing and transduction is an essential biological process for cell adaptation and survival. Histidine kinases (HK) are the sensory proteins of two-component systems that control many bacterial responses to different stimuli, like environmental changes. Here, we focused on the HK DesK from Bacillus subtilis, a paradigmatic example of a transmembrane thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C. DesK provides a tractable system for investigating the mechanism of transmembrane signaling, one of the majors interrogates in biology to date. Our studies demonstrate that transmembrane proline residues modulate the conformational switch of a 2-helix coiled-coil (2-HCC) structural motif that controls input-output in a variety of HK. Our results highlight the relevance of proline residues within sensor domains and could inspire investigations of their role in different signaling proteins.

Keywords: histidine kinase; proline; thermosensing; two-component regulatory systems.

FIG 1

Alignment of the TM region of thermosensors homologous to DesK. Sequence alignment of the transmembrane helices (TM) and the 2-helix coiled coil (2-HCC) of B. subtilis DesK, YvfT, and BA5598 of B. anthracis was produced using T-Coffe software. Conserved proline residues studied in this work are shown in orange. Leu174 is highlighted in yellow. a and d indicate positions of the heptad repeats. The positions that stabilize the 2-HCC by hydrophobic interactions are in black.

FIG 2

Pattern of Pdes-lacZ expression in the wild type and DesKPA mutants. Plasmids expressing different DesKPA mutants (DesK_P16A, DesK_P26A, DesK_P135A, and DesK_P148A) or wild-type DesK (WT) from the xylose-inducible promoter Pxyl, as well as with empty vector (EV), were transformed in the reporter strains, as indicated. (A) DAK3 transformants were streaked in LB plates in the presence of 60 μg/ml X-Gal and 0.05% xylose and incubated at 37°C (left) or for 5 h at 37°C and then transferred to 25°C for 24 h (right). (B) AKP20 transformants were grown in LB at 37°C in the absence (black bars) or presence (gray bars) of 0.05% xylose. Shown values correspond to the β-galactosidase activity of aliquots taken after 6 h of growth, expressed as a percentage of wild-type activity in the absence of xylose.

FIG 3

DesKPA_L174P double mutants respond to temperature. Plasmids expressing each of the DesKPA_L174P double mutants (DesK_P16A-L174P, DesK_P26A-L174P, DesK_P135A-L174P, and DesK_P148A-L174P), the DesKPA single mutants (DesK_P16A, DesK_P26A, DesK_P135A, and DesK_P148A), DesK_L174P, or wild-type (WT) DesK from the xylose-inducible promoter Pxyl, as well as with the empty vector (EV), were transformed in the reporter strains as indicated. (A) DAK3 transformants were grown in LB in the presence of 0.05% xylose at 37°C. At an OD₅₂₅ of 0.3 the cultures were divided into two fractions. One of them was transferred to 25°C (black bars), and the other one was kept at 37°C (gray bars). Shown values correspond to the β-galactosidase activity of aliquots measured 4 h after cold shock, expressed as a percentage of wild-type activity at 25°C. (B) AKP20 transformants were grown in LB at 37°C in the absence (black bars) or presence (gray bars) of 0.05% xylose. Shown values correspond to the β-galactosidase activity of aliquots measured after 6 h of growth, expressed as a percentage of wild-type activity in the absence of xylose.

FIG 4

Effect of L174P replacement on DesKPA variants containing two Pro-to-Ala mutations. (A) Kinase activity assay of DAK3 cells expressing the indicated DesK variants that were grown in LB in the presence of 0.05% xylose at 37°C. At an OD₅₂₅ of 0.3, the cultures were divided into two fractions. One of them was transferred to 25°C (black bars), and the other one was kept at 37°C (gray bars). Shown values correspond to the β-galactosidase activity of aliquots measured 4 h after cold shock, expressed as a percentage of wild-type activity at 25°C. (B) Phosphatase activity assay of AKP20 cells expressing the indicated DesK variants grown in LB at 37°C in the absence (black bars) or presence (gray bars) of 0.05% xylose. Shown values correspond to the β-galactosidase activity of aliquots taken after 6 h of growth, expressed as a percentage of wild-type activity in the absence of xylose.

FIG 5

Pattern of Pdes-lacZ expression in different DesK variants. Strain DAK3 was transformed with plasmids expressing wild-type DesK (WT) or different DesK alleles under the control of the xylose-inducible promoter Pxyl, as well as with the empty vector (EV). The resulting strains were streaked in LB plates in the presence of 60 μg/ml X-Gal and 0.05% xylose and incubated at 37°C (left) or for 5 h at 37°C and then transferred to 25°C for 24 h (right).

FIG 6

Destabilizing effect of Pro174. (A and B) X-ray structure of DesKC in kinase-like (PDB entry 3GIE) (A) and phosphatase (PDB entry 3EHJ) (B) states, with Leu174 rendered as spheres to highlight its location in the 2-HCC. (C) Instability introduced by mutation L174P on the 2-HCC in a model of TM5-DesKC-P148A/L174P in the phosphatase state; notice the breaks just upstream of Pro174. (D) Hydrated state observed in an molecular dynamics simulation of wild-type TM5-DesKC in the phosphatase state; notice the slight opening around Pro148 (from our previous work [17]). (E) Compact state as observed upon molecular dynamics simulation of TM5-DesKC with only a P148A substitution; notice the more compact 2-HCC and compare to that of TM5-DesKC_P148A-L174P in panel C and in Fig. S1 at https://doi.org/10.5281/zenodo.3523363. (F) Compact state as observed upon molecular dynamics simulation of TM5-DesKC with 3 substitutions that stabilize the 2-HCC in the phosphatase state (DesK_STA from our previous work [17]). Notice an effect similar to that seen in panel E.

FIG 7

Proline insertions at internal sites of 2-HCC also restore kinase activity of DesK_P148A. (A) DesK’s DHp domain and 2-HCC linker in the phosphatase state (PDB entry 3EHJ). Mutagenized residues are highlighted: pink spheres represent those amino acids occupying d positions, and, thus, participating in the stabilization of the 2-HCC, as L174 (blue spheres); cyan spheres represent residues located on the opposite side of the helix. (B) β-Galactosidase activity of strain DAK3 transformed with plasmids expressing wild-type DesK (WT), DesK_P148A, DesK_P148A-L160P, DesK_P148A-A167P, DesK_P148A-A172P, or DesR_P148A-L174P from the xylose-inducible promoter Pxyl. Strains were grown in LB in the presence of 0.05% xylose at 37°C. At an OD₅₂₅ of 0.3, the cultures were divided into two fractions. One of them was transferred to 25°C (black bars), and the other one was kept at 37°C (gray bars). Shown values correspond to the β-galactosidase activity of aliquots measured 4 h after cold shock, expressed as a percentage of wild-type activity at 25°C.

FIG 8

Effect of 2-HCC destabilization on DesK_P148A mutant. β-Galactosidase activity of strain DAK3 transformed with plasmids expressing wild-type DesK (WT) or DesK_DEST or DesK_P148A-DEST, from the xylose-inducible promoter Pxyl. Strains were grown in LB in the presence of 0.05% xylose at 37°C. At an OD₅₂₅ of 0.3, the cultures were divided into two fractions. One of them was transferred to 25°C (black bars), and the other one was kept at 37°C (gray bars). Shown values correspond to the β-galactosidase activity of aliquots measured 4 h after cold shock, expressed as a percentage of wild-type activity at 25°C.

FIG 9

(A) Representation of MS-DesK minimal sensor. The extracellular N-terminal region of DesK is depicted in white. The single chimeric TM segment of MS-DesK is composed of the first 10 residues of DesK’s TM helix 1 (light blue) fused to the last 14 residues of TM helix 5 (pink). The adjacent 2-HCC and catalytic DesKC domain are shown in gray. The conserved prolines Pro16 and Pro148 are highlighted in red and Leu174 in yellow. (B) Effect of P148A and L174P mutations on DesK minimal sensor kinase activity. Strain DAK3 was transformed with plasmids expressing different MS-DesK alleles (MS, MS_P148A, MS_P148A-L174P, and MS_L174P) under a xylose-inducible promoter. The resulting strains were grown in LB in the presence of 0.05% xylose at 37°C. At an OD₅₂₅ of 0.3 the cultures were divided into two fractions. One of them was transferred to 25°C (black bars), and the other one was kept at 37°C (gray bars). Shown values correspond to the β-galactosidase activity of aliquots measured 4 h after cold shock, expressed as a percentage of MS-DesK activity at 25°C.