Function of lipase-specific foldase for folding and secretion of a lipase from Pseudomonas aeruginosa
Final Report 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 (KD = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium Pseudomonas 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. 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 sLifY99A, 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 sLifY99A in the complex as determined by polarization-resolved fluorescence spectroscopy. NMR solution structures of P. aeruginosa MD1 and variant MD1Y99A reveal a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLifY99A. Molecular dynamics (MD) 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. Next, we studied sLif with the three domains MD1, EHD, and MD2, which form a flexible α-helical scaffold embracing LipA in a headphone-like structure, by MD simulations in combination with NMR and fluorescence spectroscopy under in vitro conditions. We show that in the free form, the α-helical structure of Lif undergoes reversible compactions and extensions on the nano- to submillisecond timescale with temporarily loses its secondary structure. Maximum entropy method-based reweighting of the MD simulations-derived conformational ensembles using FRET restraints allowed us to identify the most populated states of free sLif, which range from partially closed to completely closed conformations. The MD data suggest that the experimentally observed compaction of free sLif minimizes the number of unsatisfied polar interaction sites resulting in similar number of polar interactions as present in the complex. The most striking difference between the free Lif ensemble and the sLif:LipA complex is their respective hydrophobic surface area. In free sLif compaction only leads to a partial burial of its hydrophobic surface whereas Lif:LipA complex formation reduces the exposed hydrophobic interface on average by about 600 Å2. Thus, a further reduction of hydrophobic surface area is the underlying principle that drives complex formation. To foster also the understanding of the mechanism of how Lif activates LipA, we applied unbiased and biased MD simulations at the atomistic level and potential of mean force computations. The results show that Lif catalyzes the activation process of LipA by structurally stabilizing an intermediate LipA conformation, particularly a β-sheet in the region of residues 17–30, such that the opening of LipA’s lid domain is facilitated. This opening allows substrate access to LipA’s catalytic site. There are several surprising and so far not fully understood aspects of that study: (1) the open state of LipA is unstable compared to the closed one according to our computational and in vitro biochemical results, (2) nearly every residue at the interface between Lif and LipA plays a role for LipA secretion in vivo but not for in vitro LipA activation. We thus speculate that further interactions of LipA with the Xcp secretion machinery and/or components of the extracellular matrix contribute to the remaining activity of secreted LipA. Altogether, our results provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain minimally structured, highly dynamic steric chaperones.
Publications
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(See online at https://doi.org/10.1038/s41467-020-19023-1) - Resolving dynamics and function of transient states in single enzyme molecules. Nature Commun. 11, e1231 (2020)
Sanabria, H., Rodnin, D., Hemmen, K., Peulen, T., Felekyan, S., Fleissner, M. R., Dimura, M, Koberling, F., Kühnemuth, R., Hubbell, W., Gohlke, H., Seidel, C. A. M.
(See online at https://doi.org/10.1038/s41467-020-14886-w) - Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation. Sci. Rep. 10, e3578 (2020)
Viegas, A., Dollinger, P., Verma, N., Kubiak, J., Viennet, T., Seidel, C. A. M., Holger Gohlke, H., Etzkorn, M., Kovacic, F., Jaeger, K.-E.
(See online at https://doi.org/10.1038/s41598-020-60093-4) - The membraneintegrated steric chaperone Lif facilitates active site opening of Pseudomonas aeruginosa lipase A. J. Comp. Chem. 41, 500-512, (2020)
Verma, N., Dollinger, P., Kovacic, F., Jaeger, K.-E., Gohlke, H.
(See online at https://doi.org/10.1002/jcc.26085)
