Hsc70 is colored in blue tones, and Hsp110 is colored in autumn tones (red, orange, yellow, brown). NBDs of Hsp110 and Hsc70 are colored yellow and light cyan, respectively. The interdomain linkers of Hsp110 and Hsc70 are red and blue, respectively. The Hsc70 SBD is dark cyan, and the Hsp110 SBDβ and SBDα are orange and brown, respectively. Subdomains IA–IIB of the NBDs are labeled in (A) and (D), and individual domains are labeled in (B); different views of the complex are related by the indicated rotations.

(A) Ribbon model of two crystallographically related Hsp110:Hsc70 heterodimers colored as in Figure 2. The dashed line demarcates the boundary between each heterodimer, but the acidic insertion loop of the Hsp110 SBDβ domain reaches across from one heterodimer to interact with the Hsc70 NBD of its neighbor.
(B) Ribbon models (colored and oriented as in [A]) of the Hsp110 (2QXL; Liu and Hendrickson, 2007) and Hsc70 (1YUW; Jiang et al., 2005) in the conformations seen when these proteins are not complexed with each other. The conformational differences between the proteins in isolation and in the nucleotide exchange complex include movement of the Hsp110 SBDα toward the Hsc70 NBD, ordering of the acidic insertion loop due to crystal packing interactions, and rotation of the Hsc70 SBD due to crystal packing and/or interactions between the interdomain linkers in the nucleotide exchange complex.

(A)–(G) and (I) show different views of the 3D reconstruction of the Hsp110:Hsc70 complex, while (H) and (J) show reconstructions of an Hsp110:Hsc70 NBD complex. (E)–(G) and (H) also show a ribbon model of the Hsp110:Hsc70 crystal complex (110 NBD: yellow; 110 SBDβ: orange; 110 SBDα: gray; 70 NBD: light cyan; 70 SBD: green) fit into the EM envelope. The model fits very well with the exception of the acidic insertion loop and the Hsc70 SBD. An alternative position for the Hsc70 SBD (red) suggested in (I) and (J) fits very well into the density that is missing in the complex in which the SBD is deleted (arrows in [G] and [H]). (K) Genetic algorithm Monte Carlo fit of the sedimentation data for the Hsp110:Hsc70 complex. Relative concentrations are shown as a color gradient (white = 0 μM, black = 16 μM). The predominant species (black center) corresponds to the dimer, while the minor species at ∼72 kDa corresponds to free Hsc70ΔC and Hsp110.

(A) Clathrin basket dissociation mediated by full-length Hsc70 and auxilin, and monitored by dynamic light scattering. The clathrin and auxilin concentrations were, respectively, 1.0 and 0.1 μM, and the Hsc70 concentration was varied as indicated. Basket dissociation was fit to y = yo + A1(exp(−t1*x) − 1) − k*x, where yo = total scatter at x = 0 s = ∼1,225,000 for 1 μM clathrin; A1 = amplitude of 1st (rapid) exponential phase; t1 = time constant of 1st exponential phase; k = rate of 2nd (slow) linear phase = decrease/s in number of scattered photons. Data were globally fit with yo held constant, since yo is determined by the constant amount of clathrin baskets added to each reaction.
(B–D) Basket dissociation reactions run with constant Hsc70 (0.2 μM), auxilin (0.1 μM), and clathrin (1 μM) concentrations but with varying concentrations of either Bag1 (B), yeast Hsp110 (Sse1; [C]), or human Hsp110 (D). Data in each panel were globally fit as in (A) with yo constant, but with A1 and t1 shared by all data sets since the model assumes that A1 and t1 are determined by the amount of Hsc70 added. Only data between 0%–80% completion of the dissociation reaction were used in fitting to reduce effects due to substrate depletion.
(E) Fractions from rapid gel exclusion chromatography of a basket dissociation reaction run with 0.6 μM clathrin (“CHC”), 0.1 μM auxilin, and 0.6 μM Hsc70.
(F) As in (E) but with 0.6 μM yeast Hsp110 (Sse1) added.

(A) The nucleotide-binding site in the Hsp110 NBD. The ADP is green, BeF₃⁻ is magenta, Mg²⁺ is brown, and residues interacting with these ligands are blue, red, yellow, and gray for positive, negative, polar, and hydrophobic. The BeF₃⁻ is bound by D8, D203, K69, and the Mg²⁺. The Mg²⁺ is also liganded by the N of N11, the N_Z of K69, and an Oβ from the ADP. Protein:ADP H bonds include the following: four between the β-phosphate and the amides of N11, N12, N13, and H206 and one to the N13 carbonyl O; two between the α-phosphate and H206 and G343 amides and one to the N13 side-chain O; three ribose O₄ and O₂ bonds to, respectively, the T344 amide and Oε₂ and N_Z of E272 and K275; five between the base N3, N9, and N6 atoms and the N_Z and carbonyl oxygens of K276 and G343 and to the Oε of Hsc70 Q33.
(B) The nucleotide-binding site in the Hsc70 NBD. Colors as in (A). Electron density from an Fo-Fc map phased with a model missing the nucleotide is shown contoured at 3.0σ. Protein:ADP H bonds include the following: one between the β-phosphate and O_γ1 of T14; two between the α-phosphate and O_Δ1 of D366 and the G202 amide; five between the ribose O₂, O₃, and O₄ atoms and the N_Z of K271, the O_ε1 of E268, and the amide and O_γ of S340; and three between the base N6 and N7 and R272 carbonyl and the O_γ of Hsp110 S32.
(C) Ribbon models of Hsp110 (yellow) and Hsc70 (cyan) NBDs with nucleotides (red) and side chains (Hsc70 Q33 and Hsp110 S32) that bridge one NBD to the adenine N6 of the nucleotide in the partner in space-filling representation.
(D) Transparent surface model of the complex with nucleotides in space-filling representation (coloring and orientation as in [C]) reveals a pore that connects the nucleotide-binding sites to the bulk solvent.
(E) Coloring of the surface according to charge (blue = positive; red = negative) reveals that the pore forms an electropositive well embedded in an electronegative surround.

(A) Superimposed ribbon models of closed ATP bound Hsc70 NBD (1KAX; Flaherty et al., 1990; orange), the NBD from a nucleotide free two-domain Hsc70 (1YUW; Jiang et al., 2005; blue), from an Hsc70 NBD:Bag1 complex (1HIX; Sondermann et al., 2001), yellow), and from the Hsp110:Hsc70 complex (light cyan). Increased opening of the NBD occurs as subdomain II swings progressively farther from IB and is greatest in the Hsp110:Hsc70 complex (the Hsc70-bound ADP from the Hsp110:Hsc70 complex is shown in red space-filling representation).
(B) Ribbon models of a closed Hsc70 NBD (orange) and free Hsp110 (light gray) superimposed on the Hsp110:Hsc70 complex (colored as in Figure 2). Selected residues forming ionic interactions (blue for positively charged; red for negative) between the proteins are in stick representation. The loops formed by residues 285–292 and 289–296 of, respectively, the Hsc70 and Hsp110 NBDs are enclosed with a dashed ellipse. Closing of the Hsc70 NBD would create steric clashes between these loops and rupture the illustrated ionic interactions.
(C) Native PAGE of complex formation between Hsp110 (aa 1–666; “110”) and either WT Hsc70DC (aa 1–554; “70”) or Hsc70ΔC E283K/D285K (“70mut”). Lanes 1–3: individual indicated proteins at 10 μM; lanes 4 and 5: indicated proteins mixed at 10 μM; lanes 6 and 7: indicated proteins mixed at 5 μM; lanes 8 and 9: indicated proteins at 2.5 μM. All reactions contained 1 mM ATP.
(D) Effects of Hsp110 on basket dissociation with either full-length WT or E283K/D285K Hsc70. Red data points: no Hsc70 added; cyan: WT Hsc70 alone; green: E283K/D285K Hsc70 alone; dark blue:WT Hsc70+0.1μM Hsp110; yellow: E283K/D285K Hsc70+0.1 μM Hsp110. Reactions were run with1 μM clathrin, 0.2 μM Hsc70, and 0.1 μM auxilin and fit as in Figure 1.
(E) Native PAGE of complex formation between Hsc70ΔC and either Hsp110 (aa 1–666) or Hsp110ΔLid (aa 1–567; “110ΔLid”). Lanes 1–3: individual indicated proteins; lane 4: Hsp70ΔC + Hsp110; lane 5: Hsc70ΔC + Hsp110ΔLid (all proteins at 10 μM).
(F) Basket dissociation reactions as in (D), but run with either full-length Hsc70 alone (yellow), Hsc70 + Hsp110 (blue), or Hsc70 + Hsp110ΔLid (green).