Significant influence of four highly conserved amino-acids in lipochaperon-active sHsps on the structure and functions of the Lo18 protein

Abstract

To cope with environmental stresses, bacteria have developed different strategies, including the production of small heat shock proteins (sHSP). All sHSPs are described for their role as molecular chaperones. Some of them, like the Lo18 protein synthesized by Oenococcus oeni, also have the particularity of acting as a lipochaperon to maintain membrane fluidity in its optimal state following cellular stresses. Lipochaperon activity is poorly characterized and very little information is available on the domains or amino-acids key to this activity. The aim in this paper is to investigate the importance at the protein structure and function level of four highly conserved residues in sHSP exhibiting lipochaperon activity. Thus, by combining in silico, in vitro and in vivo approaches the importance of three amino-acids present in the core of the protein was shown to maintain both the structure of Lo18 and its functions.

Figure 1

(A) Sequence alignment of Lo18 or modified proteins. The alignments are performed on the α-crystallin domain of O. oeni Lo18 (accession number CAA67831) or modified after point substitution. The following E60K, T79V, G82V and R99D modifications are indicated with arrows below the sequences. The alignment was performed with MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/). Stars, double points and points indicate amino acid residues that are identical in 100%, 80% and 60%, respectively, of all the proteins. (B–K) Measurement of the fluorescence anisotropy of DPH inserted into O. oeni liposomes during a temperature rise between 16 and 64 °C (B–F) or after a thermal shock at 45 °C (G–K). Each panel illustrates liposome fluidisation in the absence of Lo18 (represented by a dotted line and white circles), in the presence of Lo18 WT (represented by a dotted line and black circles), or modified variants of Lo18, namely E60K (red circles) in panels B and G, T79V (yellow diamonds) in panels C and H, G82V (green triangles) in panels D and I, and R99D (blue squares) in panels E and J. Lysozyme (represented by purple diamonds) in panels F and K was used as a negative control in this experiment. For heat shock at 45 °C (G–K), three phases were defined: the equilibration phase (before 0 min), in which the reaction mixture consists of liposomes and the DPH probe in buffer, the addition of sHSPs (represented by a solid arrow) and the application of heat shock at 45 °C (dotted arrow). The data represent the means and SEs of three independent experiments.

Figure 2

Thermostabilization of proteins from E. coli cellular lysates were surproduction of Lo18 or modified protein are induced. A E. coli construction with empty plasmid has been used as negative controle (C−). The protein concentration of the lysate was fixe at 3.5 mg/mL, then heat 30 min at 55 °C. The amount of protein unaggregated were measured. The data represent the means and SE of three independent experiments.

Figure 3

Protein structure modifications induced by point mutation by in silico measurement. Impact of E60K, T79V, G82V and R99D amino acid substitution on predicted 3D Lo18 structure. Substituted amino acids are represented in red, neighbor amino acids able to interact with the initial residues in blue and neighbor amino acids able to interact with substituted residues in green. Interactions between amino acids are represented by yellow dotted line.

Figure 4

Measuring the impact of point modifications on the structure of proteins. In silico prediction of secondary structure of the modified protein by PredictPortein (A)Structure modification of Lo18 (black), E60K (red), T79V (yellow), G82V (green) and R99D (blue) induced by thermal ramping (25 °C to 50 °C on the four types of secondary structures, (B) α-helix, (C) β-sheet, (D) turns and (E) other secondary structures, evaluated by SRCD. The data represent the means and SE of three independent experiments. (F) Prediction of the impact of point mutation substitution on secondary structure accessibility. (G) Oligomeric structure of Lo18 WT or modified. Immunolabelling of Lo18 after in vivo cross-linking. The diamond-shaped numbers from 1 to 3 indicate the presence of monomer, dimer and trimer respectively.

Figure 5

Impact of Lo18/liposomes interaction on protein secondary structure. (A) Structure modification of Lo18 WT induce by thermal slope (20 °C to 60 °C) on the four main secondary structures α-helix, β-sheet, turns and other secondary structure in presence (black square) or absence (grey dot) of liposomes. The data represent the means and SE of three independent experiments. (B) SRCD spectra on 190–210 nm wavelenght for Lo18 WT (black), E60K (red), T79V (yellow), G82V (green) and R99D (blue) in presence (dark square) or absence (light dot) of liposomes at 25 °C.