Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Abstract

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.

Keywords: chloroplast; isothermal titration calorimetry (ITC); membrane transport; molecular dynamics; protein dynamic; protein targeting; signal recognition particle (SRP); single molecule biophysics.

FIGURE 1.

a, the arrangement of domains in cpSPR43. b, the amino acid positions used for fluorescence labeling highlighted in red for five double Cys cpSRP43 proteins (labeled P1–P5). The structure for the CD1-Ank1–4-CD2 region is taken from Protein Data Bank code 3UI2, and the structure for CD3 is taken from Protein Data Bank code 1X3P. c, LHCP integration assays confirming that fluorescently labeled cpSRP43 is able to transport and integrate LHCP into thylakoid membranes. TP, translation product; +, positive control with native cpSRP43/54; −, negative control with no cpSPR43/54; Ank2, WT cpSRP54 + Ank2 labeled cpSRP43; Ank4, WT cpSRP54 + Ank4 labeled cpSRP43; CD2, WT cpSRP54 + CD2 labeled cpSRP43; CD3, WT cpSRP54 + CD3 labeled cpSRP43.

FIGURE 2.

Fluorescence correlation spectroscopy curves of full-length (a) and truncated (trunc) (b) cpSRP43 in the absence (red lines) and presence of 100 nm (green lines) and 3 μm (blue lines) of cpSRP54 are shown. Titration curves of cpSRP54 binding to full-length (c) and truncated (d) cpSRP43 show that the affinity is not affected by either labeling or truncation of cpSRP43. ACF, autocorrelation function.

FIGURE 3.

smFRET histograms of Pro₁₀ and Pro₂₀ before (a) and after (b) zero FRET peak subtraction, and smFRET histograms of cpSRP43 in the absence and presence of cpSRP54 before (c) and after (d) zero FRET peak subtraction are shown. The overlay of smFRET histograms of cysteine-free mutant of cpSRP43 with double cysteine mutants labeled in the CD2-CD3 domains (P4) (e) and the Ank2-CD2 domains (P3) (f) highlights the minimum and maximum nonspecific labeling. The cysteine-free smFRET histograms are shown without (red) and with (blue) bound cpSRP54. Polarization anisotropy measurements of Alexa Fluor 488 dye free in solution as well as for cpSRP43 proteins labeled with Alexa Fluor 488 in each domain without (g) and with (h) bound cpSRP54 are shown.

FIGURE 4.

a, smFRET histograms of cpSRP43 proteins without (red) and with (blue) bound cpSRP54. The expected Poisson noise-limited smFRET histograms from the average FRET based on the SAXS structure are overlaid (gray). Insets are schematic representations of the labeled proteins. b, calculated difference histograms of the cpSRP43 proteins after binding cpSRP54.

FIGURE 5.

Single molecule fluorescence traces from donor (green) and acceptor (red) dyes and their respective time-resolved FRET efficiency traces (magenta) for PEG-surface immobilized cpSRP43 proteins. a and d, P4; b and e, P4Δ; c and f, P3. Fl. Int., fluorescence intensity.

FIGURE 6.

a, distance between the labeled residues on the Ank2 and CD2 domains (P3) as a function of time from all-atom simulations in the absence (red) and presence (blue) of cpSRP54_pep. b, overlay of the all-atom structure with the SBCG model (spheres). c, the three main conformations identified from the SBCG simulations, extended, open, and closed. The closed structure for both the unrestricted (cpSRP54-unbound) and the restricted (cpSRP54-bound) simulations are shown. d, distances between the labeled residues of cpSRP43 probed in P1 (Ank2-CD3), P3 (Ank2-CD2), and P4 (CD2-CD3) during open-to-extended or open-to-closed transitions.

FIGURE 7.

a, CD1-Ank4 region of cpSRP43 highlighting the relationship between the FRET labels in P2 and P5 with groove 1 to which the L18 motif binds and groove 2. b, root mean square fluctuations (RMSF) in the Cα atoms of residues in the Ank2-Ank4 region in the absence (red) and presence (blue) of cpSRP54_pep. The L18 and cpSRP54_pep interaction residues on cpSRP43 are shown on the x axis. c, smFRET histograms for P2 in the absence (red) and presence of different concentrations of cpSRP54_pep (green, 100 nm; orange, 100 μm). No significant difference between adding 100 nm or 100 μm cpSRP54_pep was observed, suggesting that the change in the smFRET of P2 is already saturated at 100 nm cpSRP54_pep. For comparison, the smFRET of P2 + 100 nm cpSRP54 protein (blue) is shown. d, ITC data for the addition of L18 to cpSRP43 in the absence (left) and presence (right) of cpSRP54_pep.