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 (KD = 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 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. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLifY99A. 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.


Supplementary Content
Figure S1 Sequence alignment of foldases from B. glumae and P. aeruginosa.   Table S4 Fluorescence anisotropy decay fit parameters sLif and sLifY99A labeled with BDP FL Table S5 pFCS fit parameters for sLif and sLifY99A Table S6 List of used bacterial strains, plasmids and oligonucleotides.

Table S7
Acquisition parameters of the spectra used for MD1 resonance assignment and structure calculation.

Table S8
Acquisition parameters of the spectra used for MD1Y99A resonance assignment and structure calculation.
Section S1 pFCS analysis Section S2 Fluorescence anisotropy decay analysis Section S3 Determination of half-inactivation temperature

Supplementary Figures
Figure S1 Sequence alignment of foldases from B. glumae and P. aeruginosa. BgLif and PaLif share 39% identical and 52% similar amino acids, shown on a black and gray background, respectively. Sequence similarity of P. aeruginosa and B. glumae TMD, VLD, MD1, EHD and MD2 is 31%, 44%, 52%, 46% and 48%, respectively. The red bars underneath the alignment indicate α-helices of the experimentally determined BgLif structure. The conserved RxxFDY(F/C)L(S/T)A foldase motif is indicated with a green frame. Numbers in front of the sequences indicate amino acid positions in the sequences.
Time t c (ns)  Table S4.
Time t c (ns)  Overall, our data, therefore, do not allow to map the binding interface reliably.       * This experiment was recorded using non-uniformly sampling (NUS) of NMR data with 20 % sampling density. The NUS schedule was generated with the Poisson gap sampling method and the spectrum was subsequently reconstructed using hmsIST 6,7 and processed with NMRpipe 8 ). All other spectra were processed with Topspin3.5 (Bruker BioSpin).

Section S1. pFCS analysis
Polarization-resolved fluorescence correlation spectroscopy (pFCS), in which fluorescence intensity fluctuations under constant excitation are measured, is also able to resolve molecular rotational motion. Auto-and cross-correlations between different polarization channels were obtained were obtained according to Felekyan et al. 9 1, 2 ( ) = 1 + (S2a) Here, the observation volume is approximated by a 3D-Gaussian volume with 1/e 2 radii in the lateral (ω0) and axial direction (z0), ttrans is the diffusion time, b1,2,3 and tb1,b2,b3 are amplitudes and times of the bunching terms, N is the particle number, a and ta are the amplitude and time of the antibunching term, S and C characterize the rotation model (see Kask et al. 2 ), brot and global are the amplitude and correlation time associated with rotational motion. As described above, factorization of the model function (eq S4) is based on the assumption of well-separated time scales for antibunching (ta  τe) and rotational correlation (ρglobal).
Polarization resolved fluorescence intensity decay histograms Fp(tc) and Fs(tc) were recorded. The fluorescence F(tc) and anisotropy decay r(tc) parameters were recovered by global fitting o the sum Fsum(tc) and difference Fdif(tc) histograms as previously described by Mockel

Section S3. Determination of half-inactivation temperature.
Half-inactivation temperature T50, i.e. the temperature at which LipA activity is reduced to 50% in a temperature-dependent lipase activity 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 by pNPP-based lipase activity assay. Data were fitted according to: where A(T) is lipase activity at temperature T, Amax is maximum enzyme activity (here 100%) and Amin is minimum enzyme activity (here 0%), δT is a rate of activity loss. Half-inactivation temperature T50 was determined to 29.0 ± 0.2°C and 2.1 ± 0.2°C -1 .