Domain interactions determine the conformational ensemble of the periplasmic chaperone SurA

Abstract

SurA is thought to be the most important periplasmic chaperone for outer membrane protein (OMP) biogenesis. Its structure is composed of a core region and two peptidylprolyl isomerase domains, termed P1 and P2, connected by flexible linkers. As such these three independent folding units are able to adopt a number of distinct spatial positions with respect to each other. The conformational dynamics of these domains are thought to be functionally important yet are largely unresolved. Here we address this question of the conformational ensemble using sedimentation equilibrium, small-angle neutron scattering, and folding titrations. This combination of orthogonal methods converges on a SurA population that is monomeric at physiological concentrations. The conformation that dominates this population has the P1 and core domains docked to one another, for example, "P1-closed" and the P2 domain extended in solution. We discovered that the distribution of domain orientations is defined by modest and favorable interactions between the core domain and either the P1 or the P2 domains. These two peptidylprolyl domains compete with each other for core-binding but are thermodynamically uncoupled. This arrangement implies two novel insights. Firstly, an open conformation must exist to facilitate P1 and P2 exchange on the core, indicating that the open client-binding conformation is populated at low levels even in the absence of client unfolded OMPs. Secondly, competition between P1 and P2 binding paradoxically occludes the client binding site on the core, which may serve to preserve the reservoir of binding-competent apo-SurA in the periplasm.

Keywords: Escherichia coli periplasmic chaperones; SurA protein; conformational dynamics; outer membrane protein biogenesis.

FIGURE 1

SurA is Monomeric in Solution. (a) The monomeric crystal structure of SurA (Protein Data Bank [PDB] ID: 1M5Y) is shown as a surface representation with its domains colored as depicted in the sequence diagram below. The flexible linkers between the domains of SurA are colored white. In this conformation of SurA, the core (N and C regions) and P1 domains are contacting each other, while the P2 domain is extended away in a structurally isolated conformation. (b) Representative sedimentation equilibrium (SE) data set collected for SurA at a total concentration of 25 μM. These data are well described by a single‐ideal species model with a molar mass equal to 43 ± 2 kDa. This agrees with the calculated molecular weight of monomeric SurA (45 kDa). These values represent the average weight obtained from fitting three independent experiments and the standard deviation of fitting (Figure S1)

FIGURE 2

Elongated models of SurA best describe small‐angle neutron scattering (SANS) curve. (a) The Guinier region of the WT SurA SANS dataset in 98% D₂O is shown, with the data shown in darker purple used to conduct the Guinier analysis. The fit for this region is shown with a gray line, with the radius of gyration obtained from the fit shown at the bottom left corner. Additional q * R _G ranges give similar values and are shown in Table S1. (b) The experimental SANS curve is shown in violet circles with error bars to reflect the standard error of the mean with respect to the number of pixels used in data averaging. The predicted scattering profiles from P1C1 and 1M5Y are shown as solid and dashed lines, respectively. (c) The reduced χ ² of each available structural model of SurA is plotted against the predicted R _G values calculated using HullRad. ³⁰ Each point is a different conformation of SurA, with reduced χ ² and R _G values listed in Table S2. The dotted vertical line indicates the R _G value obtained from the Guinier analysis, with the error represented by the shaded gray region. The horizontal dashed line marks a cutoff for a reduced χ ² of 2. The data points for P1C1 and 1M5Y are shown in black and labeled. (d) The P1C1 structural model is shown as a surface representation and colored as described in Figure 1

FIGURE 3

Chemical denaturation titrations of SurA domain‐deletion constructs. Circular dichroism signal at 222 nm was monitored as a function the concentration of urea to measure the equilibrium unfolding of SurA constructs of varying compositions. The cartoons in the upper left‐hand corner of each plot indicate the domain organization of each construct. Each SurA construct was found to cooperatively unfold and were fit to a two‐state, linear extrapolation model. The folding stabilities of each construct are shown in the bottom right‐hand corner of each plot and are the average of three independent titrations, with errors representing the standard deviation between the three measured stabilities. The WT SurA and SurA∆P2 titrations were best fit with an m value of unfolding equal to 1.78 and the SurA∆P1 and core domain titrations were best fit with an m value of 1.75, as determined by globally fitting the three titrations for each construct separately

FIGURE 4

Thermodynamic cycle analysis reveals competitive interactions between the peptidyl prolyl isomerase (PPIase) domains and the core. Thermodynamic cycle describing the difference in stabilities between two domain deletion constructs of SurA is shown. The four SurA constructs are shown as cartoons with the most favorable conformation of SurA colored solid and transparent domains to indicate the flexibility of the P1 and P2 domains relative to the core domain. Each side of the cycle is labeled to indicate the corresponding ∆G^o in Table S3. The two indirect thermodynamic paths from WT SurA to the core domain are indicated with purple arrows, and the direct path is indicated with the diagonal arrows in the middle of the cycle. This analysis reveals that the P1 and P2 domains compete for binding to the core domain in a thermodynamically uncoupled manner. This mechanism necessitates three conformations of SurA

FIGURE 5

The relative populations of the three conformations of SurA. Cartoon models of each conformation is shown above the bar. The percent population of each conformation in solution was calculated using $∆G_{\mathrm{P}1,\mathrm{int}}^{\mathrm{o}}$ and $∆G_{\mathrm{P}2,\mathrm{int}}^{\mathrm{o}}$