Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins

Abstract

In Gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archaeal prefoldins and the mitochondrial Tim chaperones, that is the α-helical "tentacles" extend from a β-strand "body" to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis, and structure modeling.

Keywords: Disordered protein; Holdase; Jellyfish-like chaperone; Outer membrane protein (OMP) transport; Periplasm; SANS; Skp; Small-angle neutron scattering.

Figure 1

Plot of the neutron scattering length density versus % D₂O in the solvent for water compared to those for protein, RNA, DNA and the components of lipids (lipid head group and CH₂), along with their perdeuterated counterparts.

Figure 2

SANS data on an absolute scale for Skp in D₂O and H₂O buffers. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging.

Figure 3

SANS data on an absolute scale for a) Skp-OmpW and b) Skp-OmpA. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging. Distance distribution functions for c) Skp-OmpW and d) Skp-OmpA. The peak values are scaled to 1.0 so that the differences in shape can be easily observed.

Figure 4

Experimental and calculated $\sqrt{\raisebox{1ex}{$I(0)$}\!\left/ \!\raisebox{-1ex}{$c$}\right.}$ vs f_D2O for a) Skp-OmpW and b) Skp-OmpA. Error bars on the experimental values represent the standard error of the mean based on three different Guinier fits.

Figure 5

Calculation of possible values for R_g(OmpW) and CM distance, D_CM, based on R_m and R_g(Skp) obtained from the Stuhrmann analysis.

Figure 6

All-atom model structures representing Skp a) in the crystal (R_g ≈30 Å) and b) in solution (R_g ≈ 33 Å).

Figure 7

Skp-OmpW model structures tested against the SANS data as described in Materials and Methods. The ball representation of the ellipsoids is for clarity. The ellipsoids were represented by non-overlapping points for SANS curve calculations, as described in Materials and Methods.

Figure 8

Model SANS curves from a) the Skp component represented by the S1, S2 and S3 all-atom structures and b) the OmpW component represented by the e21, e24 and e27 ellipsoids. I(0) is arbitrarily scaled to 1.0.

Figure 9

Several views of Skp-OmpW model S3e24, which was the best-fit model for this complex. The green balls are at the CM locations of S3 Skp and the e24 ellipsoid. The ball representation of the ellipsoids is for clarity. The ellipsoids were represented by non-overlapping points for SANS curve calculations, as described in Materials and Methods.

Figure 10

Skp-OmpW SANS data on an absolute scale (points) along with model SANS curves (solid lines) from S3e24. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging.

Figure 11

Skp-OmpA starting structure models for SASSIE runs as described in Materials and Methods. The ball representation of the ellipsoids is for clarity. The ellipsoids were represented by non-overlapping points for SANS curve calculations, as described in Materials and Methods.

Figure 12

χ² versus R_g plots as a function of contrast from SASSIE runs exploring the conformation space of the OmpA PP domain for models a) S3e24PP4 and b) S3e24PP5.

Figure 13

χ²(avg) versus R_g(avg) plots from the plots in Figure 12 for models S3e24PP4 and S3e24PP5

Figure 14

χ² versus R_g plots as a function of contrast from SASSIE runs exploring the conformation space of the OmpA PP domain for model S3-OmpA.

Figure 15

χ²(avg) versus R_g(avg) plots from Figure 13 for S3-OmpA.

Figure 16

Model SANS data calculated from the best-fit and worst-fit single S3e24PP5 structures, as well as the average SANS curve calculated from the entire S3e24PP5 ensemble, along with the SANS data on an absolute scale for Skp-OmpA in a) 0 % D₂O, b) 15 % D₂O, c) 30 % D₂O and d) 98 % D₂O. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging.

Figure 17

Model SANS data calculated from the best-fit and worst-fit single S3-OmpA structures, as well as the average SANS curve calculated from the entire S3-OmpA ensemble, along with the SANS data on an absolute scale for Skp-OmpA in a) 0 % D₂O, b) 15 % D₂O, c) 30 % D₂O and d) 98 % D₂O. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging.

Figure 18

a) Skp-OmpA SANS data on an absolute scale along with model SANS curves from the single best-fit S3e24PP5 structure. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging. The b) best-fit and c) worst-fit model structures are also shown for comparison. The ball representation of the ellipsoids is for clarity. The ellipsoids were represented by non-overlapping points for SANS curve calculations, as described in Materials and Methods.

Figure 19

a) Skp-OmpA SANS data on an absolute scale, along with model SANS curves from the single best-fit S3-OmpA structure. Error bars represent the standard error of the mean with respect to the number of pixels used in the data averaging. The b) best-fit and c) worst-fit model structures are also shown for comparison.

Figure 20

Structure density plots representing all of conformation space (grey) and the best-fit conformation space within the rectangles in Figures 13 and 15 (green) explored by the OmpA PP domain using the S3e24PP5 and S3-OmpA models. The OmpA TM domain is shown in red and Skp in shown in black.