A molecular mechanism of chaperone-client recognition

Abstract

Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature.

Keywords: Chaperone protein; Im7; NMR spectroscopy; protein dynamics; protein folding; protein-protein interaction; spy.

Fig. 1. Interaction surface mapping of the Spy-Im7 complex.

(A) Sedimentation analysis of the Spy-Im7 complex. Signal-weighted averaged s values (s_w) for the faster sedimenting peak (black circles) were fitted with either a 2:1 or a 2:2 Spy-Im7 association model. (B) Binding of Spy to immobilized Im7 in an SPR assay. Saturation SPR response units are plotted as a function of Spy concentration. Data (points) were fitted with a 1 dimeric spy–to–1 monomeric Im7 binding model. (C) Magnitude of normalized CSP for residues M46, E58, and E125 of 200 μM dimeric Spy plotted as a function of the concentration of Im7 and fitted with the K_D obtained from SPR. (D) Two-dimensional [¹⁵N,¹H]-TROSY spectra of 200 μM dimeric WT Spy in the absence (black) and in the presence of 200 μM Im7 (cyan). (E) CSPs of amide moieties of 200 μM dimeric Spy after binding 200 μM Im7, plotted against the Spy amino acid residue number. The secondary structure of the native-state Spy is indicated by the blue bar on the top of the chart. (F) Structural representation of the CSPs mapped on the surface of Spy [from Protein Data Bank ID code 3O39 (14)]. A gray-to-cyan color scale is applied according to the magnitude of the CSPs for the interaction with Im7. The brightest cyan indicates the largest CSP.

Fig. 2. Recognition sites and dynamics of the client Im7 upon binding Spy.

(A) Two-dimensional [¹⁵N,¹H]-HSQC (heteronuclear single-quantum coherence) spectra of 200 μM Im7 titrated with increasing concentration of Spy: 0 (black), 60 (magenta), and 240 μM (orange). (B) Weighted average CSPs of amide moieties plotted against the amino acid residue number of 200 μM Im7 upon interaction with 240 μM dimeric Spy. The secondary structure of Im7 is indicated on top. Gray shades denote the strongest interacting segments. (C) Projection of the CSPs of WT Im7 upon binding with Spy on the crystal structure of Im7 [from Protein Data Bank ID code 1AYI (39)]. A gray-to-orange color scale is applied according to the magnitude of the CSPs for the interaction with Spy. The brightest orange indicates the largest CSP. (D) Local frustration of Im7 is depicted on its crystal structure (39). Clusters of maximum and minimum frustrated contacts are shown by red and green dash lines, respectively. (E and F) Plots of ¹⁵N-{¹H} heteronuclear NOE of apo Im7 (green) and holo Im7 (orange). (G) The subtraction of the ¹⁵N-{¹H} heteronuclear NOE of apo Im7 with the ¹⁵N-{¹H} heteronuclear NOE of holo Im7. (H) C_α-C_β secondary chemical shift deviation changes between apo and holo forms of Im7. (I and J) Percentage of the amide protons of the apo (I) and holo forms (J) of Im7 that exchanged with deuterium within 3 min.

Fig. 3. Spatial organization of the Spy-Im7 complex.

(A) Distance restraints from intermolecular PRE measurements relative to a value of 15 Å. Data of four single mutants of Spy attached with the spin label MTSL are shown: yellow, Spy T72C; orange, Spy M53C; blue, Spy T99C; violet, Spy T123C. Residues 23 to 36 and 50 to 65, which have an averaged distance of less than 15 Å to the spin label center of M53C, M85C, T99C, and T123C mutants, are indicated with green background. Right: For each spin label, spheres with a radius of 20 Å centered around the C_β atom of each cysteine mutant are shown on the crystal structure of Spy. (B) Rigid-body docking model of the complex of Spy with Im7, based on the PRE data. Three representative ensemble members are shown.

Fig. 4. Compaction effect of the Im7_U ensemble by Spy.

(A) Two-dimensional ¹⁵N-¹H HSQC spectra of 200 μM Im7_U [Im7(L18A,L19A,L37A)] in the absence (black) and in the presence of 200 μM dimeric Spy (violet). (B) Display of the CSPs of Im7_U upon binding with Spy on a random coil model of Im7_U. (C) Two-dimensional [¹⁵N,¹H]-TROSY spectra of 200 μM dimeric WT Spy in the absence (black) and in the presence of 200 μM Im7_U (red). (D) Structural representation of the CSPs mapped on the surface of Spy [from Protein Data Bank ID code 3O39 (14)]. The brightest red indicates the largest CSP. (E and F) The weighted average CSPs of amide moieties of Im7 and Spy upon binding each other are calculated and plotted against the Im7 and Spy amino acid residue number. The secondary structures of both Im7 and Spy are indicated by the blue bar on top of the chart. (G) Intermolecular paramagnetic relaxation effect on ¹⁵N-labeled Im7_U upon interaction with the spin label MTSL–attached M85C Spy, calculated as I_ox/I_red, the ratio of the heights of peaks before and after the reduction of the spin label. (H and I) Compactness of Im7_U in the absence and presence of Spy. Intramolecular PRE effect of a paramagnetic spin label (MTSL) attached to Im7_U at positions T30C (H) and T11C (I). (H) Top: Data for T30C in the absence of Spy. Bottom: Data for T30C in the presence of Spy. (I) Top: Data for T11C in the absence of Spy. Bottom: Data for T11C in the presence of Spy.

Fig. 5. The interaction sites on Im7 and Im7_U for the chaperones Spy, SurA, and Skp.

CSPs of amide moieties plotted against the amino acid residue number of 200 μM Im7 (left column) and 200 μM Im7_U (right column) upon interaction with any of the chaperones 240 μM dimeric Spy (top row), 200 μM SurA (central row), or 200 μM trimeric Skp (bottom row). The secondary structure elements H1 to H4 of Im7 are indicated. Segments 19 to 41 and 55 to 66 are highlighted in gray to guide the eye. Structural models are shown for orientation (40, 41). Data in top row are identical to Figs. 2B and 4F.

Fig. 6. Model of chaperone Spy in recognition, interaction, and release of the client Im7.

Unfolded client Im7 (Im7_U, purple) is recognized and held in a compacted ensemble state by the chaperone Spy. The client Im7 becomes more dynamic when bound to Spy. Partially folded Im7 binds with its locally frustrated segment to Spy. Client release is achieved by propagation toward a fully folded client, which in the case of Im7 can be achieved by binding to its natural complex partner colicin E7.