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

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 ( s Lif) that isolated MD1 inhibits s Lif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant s Lif 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 s Lif 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 s Lif Y99A . Molecular dynamics simulations of the s Lif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of s Lif 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.