Two-step mechanism of J-domain action in driving Hsp70 function.

J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface-thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition-via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage

Structure and evolution of the 4-helix bundle domain of Zuotin, a J-domain protein co-chaperone of Hsp70.

The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserved in eukaryotes. However, whether the 4-helix bundle (4HB) domain is conserved remains an open question. We undertook evolutionary and structural approaches to clarify this issue. We found that the 4HB segment of human Zuotin also forms a bundle of 4 helices. The positive charge of Helix I, which in Saccharomyces cerevisiae is responsible for interaction with the 40S subunit, is particularly conserved. However, the C-termini of fungal and human 4HBs are not similar. In fungi the C-terminal segment forms a plug that folds back into the bundle; in S. cerevisiae it plays an important role in bundle stability and, off the ribosome, in transcriptional activation. In human, C-terminal helix IV of the 4HB is extended, protruding from the bundle. This extension serves as a linker to the regulatory SANT domains, which are present in animals, plants and protists, but not fungi. Further analysis of Zuotin sequences revealed that the plug likely arose as a result of genomic rearrangement upon SANT domain loss early in the fungal lineage. In the lineage leading to S. cerevisiae, the 4HB was subjected to positive selection with the plug becoming increasingly hydrophobic. Eventually, these hydrophobic plug residues were coopted for a novel regulatory function-activation of a recently emerged transcription factor, Pdr1. Our data suggests that Zuotin evolved off-ribosome functions twice-once involving SANT domains, then later in fungi, after SANT domain loss, by coopting the hydrophobic plug. Zuotin serves as an example of complex intertwining of molecular chaperone function and cell regulation

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system

<div><p>By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on <i>Saccharomyces cerevisiae SIS1</i>, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking <i>SIS1</i> to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if <i>YDJ1</i> suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for <i>SSA1</i> suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70’s ability to function in a wide range of cellular processes—modulating the Hsp70-substrate interaction cycle.</p></div

Substitutions in Ssa1 allow growth of <i>sis1-Δ</i> cells.

<p>(<b>A,B</b>) Ten-fold serial dilutions of <i>sis1-Δ</i> strains harboring a plasmid with an insert of a WT <i>SIS1</i> gene (<i>SIS1</i>), the originally isolated <i>SSA1</i> suppressor mutant (<i>ssa1</i>_<i>SUP</i>) or the indicated <i>SSA1</i> substitutions. Plates were incubated at indicated temperatures for 4 days. (<b>C</b>) <i>sis1-Δ</i> cells harboring a plasmid with a WT <i>SIS1</i> gene and <i>URA3</i> marker plus the indicated additional <i>TRP1</i>-marked plasmid were streaked onto plates containing 5-FOA and incubated for 5 days at 30°C. <i>TRP1</i> plasmids had inserts encoding WT (<i>SIS1</i>), or <i>SSA1</i> genes, either WT (<i>SSA1</i>) or indicated variants. ΔEEVD indicates that the C-terminal four residues are absent.</p

Substitutions of Y66 in helix IV of the J-domain allow Ydj1₁₃₄ to support growth of <i>sis1-Δ</i>.

<p>(<b>A</b>) (<i>left</i>) Cells lacking <i>SIS1</i> (<i>sis1-</i>Δ), harboring a plasmid carrying an insert encoding WT Sis1 (<i>SIS1</i>), or Ydj1₁₃₄ with no substitutions (WT) or indicated substitution, were plated as 1:10 serial dilutions and grown at 30 or 34°C for 2 days, or at 37°C for 3 days. (<i>right</i>) Lysates prepared from the strains were subjected to immunoblotting using antibodies against the J-domain of Ydj1 or Ssc1 (control). (<b>B</b>) Diagram of Ydj1 J-domain/Gly-rich region with sequence and position of helices in segment encompassing residues 50–85 indicated; suppressor positions Y66 and G70 in red. (<b>C</b>) Averaged NMR structure of Ydj1 J-domain (N-terminal 70 residues) generated from coordinates of 20 lowest-energy conformers presented as ribbon representation and colored by secondary structure (gray for α-helix, green for unstructured regions). (<b>D</b>) (<i>left</i>) Ydj1 J-domain structure with Y66 and G70 (red) and residues with which Y66 interacts (yellow; F7, Y8, I56, L57) in sphere representation. (<i>right</i>) Most affected residues (i.e. those with the largest CSPs (red) and those missing signals in Ydj1₁₀₉G70N (blue) as shown in Fig 3C) mapped on Ydj1 J-domain structure and in the sequence of the Gly-rich region.</p

G70N substitution in Ydj1 affects structural properties around the junction of J-domain and Gly-rich region.

<p>(<b>A</b>) Single turnover ATPase assays were performed using ³²P-ATP/Ssa1 complex. Data are presented as fold-stimulation at indicated ratios of Ydj1 or Ydj1G70N, relative to basal Ssa1activity. Error bars indicate the standard deviation. The mean of 6 experiments is plotted. (<b>B</b>) (<i>left</i>) 10-fold serial dilutions of cells lacking Sis1 (<i>sis1-</i>Δ) carrying a plasmid with an insert encoding WT Sis1 (<i>SIS1</i>) or the 109 or 134 residue N-terminal Ydj1 fragment with the G70N substitution (<i>ydj1</i>_<i>109</i><i>G70N</i> and <i>ydj1</i>_<i>134</i><i>G70N</i>, respectively). (<i>right</i>) Lysates made from these strains were subjected to immunoblot analysis using antibodies specific for the Ydj1 J-domain and, as a control, Ssc1. (<b>C</b>) NMR analysis of conformational changes in Ydj1₁₀₉ due to G70N substitution. (<i>top</i>) Comparison of 2D ¹H,¹⁵N HSQC spectra obtained for Ydj1₁₀₉WT (black) and Ydj1₁₀₉G70N (green). The signal position of each residue is defined by chemical shifts along the ¹⁵N (y-axis) and ¹H (x-axis) dimension, which reflects protein conformation. (<i>right</i>) Overlay of the two HSQC spectra. Most signals are very similar, with a number of signals exhibit substantial differences in position. Arrows indicate signals of the 8 most affected residues: WT (solid), G70N (dotted). (<i>bottom)</i> Histogram representing differences in each residue’s signal position in Ydj1₁₀₉WT and Ydj1₁₀₉G70N HSQC spectra. Change indicated by height of bar, calculated as the combined difference in chemical shifts between signals for each residue. The magnitudes of the chemical shift perturbations (CSP) are color-coded: (red) CSP >0.1 ppm; (gray) CSP ≤ 0.1 ppm; (blue rectangles) signals from residues T5-D9 and L57-Q68 that were identified in the Ydj1₁₀₉WT spectrum, but not in Ydj1₁₀₉G70N; (star) proline residues, which have no signal in HSQC spectra. The α-helices of the J-domain are indicated at the top of the panel. The G70N substitution is indicated by a dotted line.</p

G70N and Y66H substitutions in Ydj1 allow maintenance of the strong [<i>PSI</i>^<i>+</i>] prion.

<p>(<b>A</b>) Maintenance of Sc4 (strong) and Sc37 (weak) [<i>PSI</i>^<i>+</i>] by Ydj1₁₃₄ suppressor variants in <i>sis1-Δ</i> strains. Equal number of cells were dropped on YPD and grown at 23˚C for 5 days for assessment of color development. Dashed line indicates different parts of the same plate where irrelevant strains were cropped out of the image. For both variants, a representative of the 10 candidates isolated from 5-FOA plates that were tested for [<i>PSI</i>^<i>+</i>] maintenance; all showed similar color phenotype. Parent [<i>PSI</i>^<i>+</i>] cells for each prion variant (parent) and cells cured of the prion by growth in the presence of GdnHCL (cured). (<b>B</b>) For analysis of [<i>PSI</i>^<i>+</i>], cell lysates were prepared, resolved by SDD-AGE and subjected to immunoblotting using Sup35-specific antibodies.</p

Isolation of a spontaneous <i>YDJ1</i> suppressor of <i>sis1-Δ</i>.

<p><b>(A)</b> Overview of Hsp70 interaction with J-domain and substrate. When Hsp70 is bound to ATP (left), substrate has easy access to the cleft in the β-subdomain of SBD (SBDβ). This is often called the open-or docked-state, because the lid (SBDα), which traps substrate by covering the cleft when ADP is bound (right), is restrained through interaction with the NBD; the SBDβ and inter-domain linker are also docked on the NBD. In concert with substrate in the cleft, J-domain binding at the NBD-SBDβ interface stimulates hydrolysis of ATP to ADP. The resulting conformational changes cause the domains to disengage, forming the undocked/closed state and stabilizing substrate interaction. Bracket indicates dynamic transitions between predominant ATP and ADP conformations with dotted line indicating movement of the SBDα lid. Nucleotide release by nucleotide exchange factors (NEF) and rebinding of ATP completes the cycle (bottom). SBDα (green), SBDβ (yellow), NBD (gray), linker (black). (<b>B</b>) <i>sis1-Δ</i> cells carrying a plasmid with a wild type (WT) <i>SIS1</i> gene and <i>URA3</i> marker, plus the indicated additional plasmid having a <i>TRP1</i> marker, and/or having the genomic suppressor mutation were streaked on plates containing 5-FOA. Plasmid with <i>TRP1</i> marker: WT copy of <i>SIS1</i> (<i>SIS1</i>) or no insert (-); on the chromosome (Chrom): SUP indicates presence of spontaneous suppressor mutation. Plate was incubated for 3 days at 30°C. (<b>C</b>) Schematic representation of Sis1 and Ydj1 architecture. J, J-domain (red); Gly, Glycine-rich region (green); CTDI/CTDII, C-terminal domains I and II, each of which is a b-barrel (blue); ZBD, zinc binding domain (yellow); DD, dimerization domain (purple). Note: the Sis1 Gly-rich region is often divided into a G/F region (residues 69–121) and the G/M region (122–177), as the former is rich in phenylalanine, and the later in methionine, as well glycine. (<b>D</b>) (<i>left</i>) Ten-fold serial dilutions of <i>sis1-Δ</i> strains with no additional chromosomal mutation (-), a deletion of <i>YDJ1</i> (<i>ydj1-Δ</i>), or the suppressor mutation (SUP) were grown at indicated temperatures for 3 days. Strains also carried a <i>TRP1</i> plasmid: no insert (-) or the <i>YDJ1</i> gene cloned from the suppressor strain (<i>ydj1</i>_<i>SUP</i><i>)</i> under the control of its native promoter or the <i>TEF2</i> promoter (superscript <i>YDJ1</i> or <i>TEF</i>, respectively). (<i>right</i>) Cell lysates of strains were subjected to immunoblot analysis using antibodies specific for the Ydj1 J-domain or, as a control, Ssc1. (<b>E</b>) (<i>left</i>) Comparison of growth of a <i>sis1-Δ</i> strain with a plasmid carrying WT <i>SIS1</i> or, under control of the <i>TEF2</i> promoter, a truncated <i>YDJ1</i> encoding the N-terminal 134 residues (<i>YDJ1</i>_<i>134</i>), either WT in sequence or encoding the indicated substitutions of residue G70. Cells were grown at indicated temperatures for 2 days. Dashed line indicates that two plates were used; cells were grown, plated and incubated side by side. (<i>right</i>) Comparison of Ydj1 levels in strains expressing <i>ydj1</i>_<i>134</i> variants; in addition, WT <i>YDJ</i>_<i>134</i> expressed in <i>ydj1-Δ</i> strain is shown as an expression control at far left. Cell extracts were subjected to immunoblot analysis using antibodies against the J-domain of Ydj1 or, as a control, Ssc1.</p

Functionality of Ssa1 suppressor variants in cells lacking WT <i>SSA</i> genes.

<p>(<b>A</b>) (<i>left</i>) Ten-fold serial dilutions of a <i>ssa1-Δ ssa2-Δ ssa3-Δ ssa4-Δ</i> (<i>ssa1-4Δ</i>) strains expressing the indicated <i>SSA1</i> gene from a plasmid: WT; original suppressor isolate (<i>ssa1</i>_<i>SUP</i>); deletion of codons for four C-terminal residues (ΔEEVD). Plates were incubated at the indicated temperatures for 2 days. (<i>right</i>) Cell lysates were subjected to immunoblot analysis to compare Ssa1 levels using antibodies raised against Ssa1 or Tim44 (control). (<b>B</b>) <i>ssa1-Δ ssa2-Δ ssa3-Δ ssa4-Δ</i> strains harboring two plasmids, one with WT <i>SSA1</i> and a <i>URA3</i> marker and a second <i>TRP1</i>-marked plasmid with no insert (-) or the indicated <i>SSA1</i> gene were plated on medium containing 5-FOA. Plates were incubated for 3 days at 30°C. (<b>C</b>) Ten-fold serial dilutions of a <i>ydj1-Δ</i> strains harboring a plasmid with no insert (-) or a <i>YDJ1</i> gene (either WT or with G70S substitution) were plated and incubated at the indicated temperatures for 2 days. (<b>D</b>) Ten-fold serial dilutions of <i>sis1-Δ</i> cells expressing the <i>Ssa1</i>_<i>K446E</i> from a plasmid, and having either WT or deletions of <i>SSA1-4</i> (<i>ssa1-4-Δ</i>) on the chromosome. After plating, cells were incubated at the indicated temperatures for either 4 days (23°C) or 3 days (30, 34°C).</p