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. 2016 Dec 21;5:e21848. doi: 10.7554/eLife.21848

Figure 1. SEC-MALS reveals a switch of LapD dimers to dimer-of-dimers upon ligand binding.

(A) Working model for c-di-GMP-dependent regulation of the periplasmic protease LapG via the inner membrane protein LapD. Concerted conformational changes expose a periplasmic binding site for LapG on LapD, sequestering the protease away from its substrates, the adhesin proteins LapA in P. fluorescens or CdrA in P. aeruginosa. (DGC, diguanylate cyclase; red/orange asterisks indicate interaction helices in LapD/GcbC). (B). Size-exclusion chromatograms for detergent-solubilized LapD in different states. Samples were prepared as described in the Material and Methods. (Asterisks: c-di-GMP was included in the mobile phase). (C) Molecular weight of LapD in solution. Peak fractions were analyzed by in-line SEC-MALS. (Absorbance at 280 nm: Traces colored according to (B); molecular weight determination: Dark and light purple dots; theoretical molecular weights based on sequence: Horizontal dashed lines.) Data are representative of two biological replicates using independent protein preparations.

DOI: http://dx.doi.org/10.7554/eLife.21848.002

Figure 1.

Figure 1—figure supplement 1. Mapping of physical interaction motifs onto the crystal structures of LapD and GcbC.

Figure 1—figure supplement 1.

Previous studies identified helical motifs in LapD’s and GcbC’s cytosolic domains (asterisks in Figure 1A) and GcbC’s I-site as features that support interactions between this receptor–DGC pair (Dahlstrom et al., 2015, 2016). Structures: LapD’s S helix-GGDEF-EAL domain, left, PDB code 3pjx; GcbC’s GGDEF domain bound to c-di-GMP, middle, PDB code 5euh; LapD’s EAL domain dimer bound to c-di-GMP, right, PDB code 3pjt).
DOI: http://dx.doi.org/10.7554/eLife.21848.003

Figure 1—figure supplement 2. C-di-GMP binding to LapD results in a 6-fold increase in apparent affinity for LapG.

Figure 1—figure supplement 2.

An equilibrium-saturation binding experiment based on fluorescence anisotropy is shown. See Material and Methods for details. Means and standard errors from two biological and two technical replicates are shown. (Minus c-di-GMP — kD = 11.7 ± 3.8 μM, Bmax = 0.1 ± 0.01; plus c-di-GMP — kD = 1.9 ± 0.3 μM, Bmax = 0.09 ± 0.004.)
DOI: http://dx.doi.org/10.7554/eLife.21848.004

Figure 1—figure supplement 3. SEC-MALS analyses of protein–detergent complexes.

Figure 1—figure supplement 3.

(A) SEC-MALS analysis of LapD incubated with LapG and c-di-GMP. In this experiment, c-di-GMP was omitted from the mobile phase. (B) Experimental determination of detergent dn/dc values. Means and standard errors from three biological replicates are shown. (C) Molecular weights associated with various states of LapD were determined by SEC-MALS. aPeaks 1 and 2 refer to the species eluting at 10–11 min and 12 min, respectively, observed in the associated chromatograms shown in Figure 1C and panel (A) of this figure. bMolecular weight of protein–detergent complex, which is the sum of the protein component molecular weight (c) and the detergent component molecular weight (d). #Theoretical molecular weights: LapD dimer = 146.0 kDa, LapD dimer with one LapG = 168.9 kDa, and LapD dimer of dimers with two LapG molecules = 337.8 kDa. *c-di-GMP present in mobile phase of SEC.
DOI: http://dx.doi.org/10.7554/eLife.21848.005