Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

Abstract

Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K_D = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLif_Y99A, in which the evolutionary conserved amino acid Y99 from helix α1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLif_Y99A in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1_Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLif_Y99A. Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.

Figure 1

Schematic representation of P. aeruginosa Lif and its complex with lipase LipA. (A) Five-domain organization of Lif and (B) Lif-LipA complex. The catalytic folding domain (CFD) self-sufficient for activation of LipA in vitro comprises MD1, EHD and MD2. Residues defining the beginning and the end of each domain are indicated in (A).

Figure 2

Activation of P. aeruginosa lipase LipA with sLif and variant sLif_Y99A and effect of MD1. (A) Pre-active LipA (4 µM) was incubated with either sLif (4 µM) or variant sLif_Y99A (4 µM) followed by lipase activity assay with 10 nM LipA. The activity of sLif:LipA complex was set as 100%. (B) SDS-PAGE analysis of LipA co-purified in the complex with sLif or sLif_Y99A as well as without Lif (w/o Lif). In the Coomassie Brilliant Blue G250 stained gel LipA is migrating as ~30 kDa and sLif as 43 kDa protein. Molecular weights of standard proteins (St) are indicated on the left-hand side. (C) Inhibition of sLif-mediated LipA activation with MD1. Pre-active LipA (50 nM) incubated with MD1 or MD1_Y99A was activated by addition of sLif (50 nM) and 10 min incubation prior to lipase activity measurement. Lipase activities are mean values ± standard deviation of three independent experiments each measured with at least three samples. (D) A fluorescence assay was used to study the complex formation of pre-active LipA and sLif/sLif_Y99A labelled at amino groups with BDP FL. The fraction of fluorescence parameters assigned to the sLif:LipA complex (steady-state anisotropy r_steady-state (Eq. 2, Table S1) and average translational diffusion time ‹t_trans› (Eq. 3, Table S2). The binding data were fitted with a 1:1 binding affinity model (Eq. 4), black line. The uncertainties are indicated as shaded areas. Steady-state anisotropy could not be used for sLif_Y99A:LipA complex because increased mobility of the fluorescent probe cancels the increase of global rotation correlation time ρ_global. The apparent dissociation constant K_D (right panel) was determined to 29 nM ± 9 nM for sLif:LipA and 77 nM ± 24 nM for sLif_Y99A:LipA complex (error bars are standard errors of the fit).

Figure 3

Effect of temperature on unfolding and activity of LipA in complex with sLif and variant sLif_Y99A. (A) Melting curves of sLif, sLif_Y99A, pre-active LipA alone and after incubation with sLif, sLif_Y99A, MD1 and MD1_Y99A obtained by fluorescence measurement with nanoDSF. (B) Temperature-dependent lipase activity in a solution of sLif and LipA generated by incubation of pre-active LipA (100 nM) with sLif (250 nM) overnight at 4 °C in TG buffer. Samples were then incubated at different temperatures (10–50 °C) for 1 h followed by measurement of the remaining lipase activity with 2 nM LipA. (C) Time-resolved fluorescence anisotropy decay curves r(t_c) of free and complex sLif and sLif_Y99A. Open circles indicate experimental r(t_c) and lines indicate model r(t_c) (Eq. S2b, results see Table S4), dashed lines indicate complex. Further details see main text. (D) Polarization-resolved full-FCS of labelled sLif using the p-p cross-correlation curves G_p-p(t_c) normalised to the number of molecules in focal volume (Eq. S3) together with weighted residuals of the fits (upper plot, Eq. S4, results in Table S5). The global rotation correlation time ρ_global of sLif is similar to the one obtained by anisotropy measurement (32 ± 3 ns, indicated by vertical line). The global rotation correlation times of sLif:LipA and sLif_Y99A:LipA are similar (50 ± 3 ns, vertical line). (E) Joint analysis of the anisotropy order parameters (solid lines, see shaded area in C) and normalized pFCS amplitudes (dashed lines, see shaded area in D) by displaying the model functions of the fits. The global rotational correlation times are depicted as vertical lines. The corresponding amplitudes are highlighted by arrows. For further details see main text.

Figure 4

Details of MD1 and variant MD1_Y99A structures obtained by NMR spectroscopy. (A) Cartoon representations of the structure ensemble of the 20 best solution structures of MD1 and (B) MD1_Y99A variant. (C) Comparison of the representative NMR solution structures of MD1 (cyan) and MD1_Y99A (purple) with the crystal structure of MD1 from B. glumae (green) (PDB code 2ES4).

Figure 5

NMR-based structural comparison of MD1 and MD1_Y99A. (A) ¹H-¹⁵N-HSCQ spectra of MD1 (black) and MD1_Y99A (red). Labels correspond to the most affected residues due to the mutation. (B) Chemical shift perturbations induced by the mutation along the MD1 sequence and (C) mapped on the MD1 structure (purple, mutation site highlighted). (D) Comparison of ¹³C secondary chemical shifts of MD1 (black) and MD1_Y99A (red). Positive/negative values indicate α-helical/β-strand secondary structure. Random coil values should be zero. (E) Inter-residue distance restraints from NOEs for MD1 (black) and MD1_Y99A (red).

Figure 6

Influence of mutation Y99A in Lif on the structural stability of LipA. (A) Structure of P. aeruginosa LipA with OCP inhibitor bound in the active site crystallized in the open conformation (PDB code 1EX9), in which the helix α5 (salmon) is moved away from the active site (catalytic triad residues S82, H251 and D229 shown in green). In this conformation, the active site is accessible for the substrate and LipA is enzymatically active. A short two-stranded β-sheet close to the active site is formed by residues 17–30 (red). (B) B. glumae lipase from the crystal structure of the Lif:LipA complex (PDB code 2ES4). The lipase shows a two-stranded β-sheet (red), a characteristic feature of the open (active) conformation, nevertheless helix α5 (salmon) adopts a closed (inactive) conformation. This suggests a foldase-induced formation of a two-stranded β-sheet during activation of the lipase. (C) Crystal structure of B. glumae lipase crystallized in the closed conformation (PDB code 1QGE) with helix α5 (salmon) covering the active site (residues as in panel A). In this conformation, a two-stranded β-sheet close to the active site is not formed. Residues 17–30 of B. glumae lipase, forming a two-stranded β-sheet, are indicated red. (D) Crystal structure of active P. aeruginosa LipA with inhibitor OCP bound in the active site (PDB code 1EX9). Region of residues 17–30 forms part of the active site (red), required for the binding of the ligand. (E) Homology model of the P. aeruginosa sLif:LipA complex based on the structure of the B. glumae foldase-lipase complex (PDB code 2ES4) used as a template. The coloring indicates the model quality assessment by TopScore^,, with bluish colors representing less than 10% structural error. (F) CNA was applied on an ensemble of structures of the Lif:LipA complex generated from 10 independent MD simulations. Residues with $\mathrm{\Delta }{G}_{i,\mathrm{CNA}}$ above a threshold of 0.1 kcal mol⁻¹ are depicted as spheres on the Lif:LipA complex structure. Blue colors reflect predicted $\mathrm{\Delta }{G}_{i,\mathrm{CNA}}$ values; the larger the value, the darker is the color. The black arrow indicates how the perturbation by Y99A mutation of Lif (pink, ball-and-stick representation) influences residues in LipA. Due to the decrease in the stability of the surrounding region of residues 17–30 in LipA, we speculate that the conformational changes required for the intermediate state of LipA on the way of activation is hampered upon Lif_Y99A mutation. The color code for helix α5, residues 17–30 and the active site is as in panel (A). (G) The histogram shows the per-residue $\mathrm{\Delta }{G}_{i,\mathrm{CNA}}$ for LipA. The dashed line at 0.1 kcal mol⁻¹ indicates the threshold above which residues are considered perturbed. The standard error of the mean is <0.05 kcal mol⁻¹ for all residues. (H) Per-residue $\mathrm{\Delta }{G}_{i,\mathrm{CNA}}$ shown for Lif, with the same threshold. The standard error of the mean is <0.05 kcal mol⁻¹ for all residues.