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* This work was supported by National Institutes of Health Grants AG045223 (to L. S.), AG031782 (to A. M. C. and L. S.), NS059690 (to E. R. P. Z. and J. E. G.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains supplemental Figs. S1–S6.
hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4–5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.
hsc-70 (HSPA8) is a constitutively expressed molecular chaperone. The human hsp-70 chaperone family consists of 11 highly homologous members specific to different cellular compartments and organelles (
(residues 1–384) with ATPase activity; a 12-kDa substrate binding domain (SBD) that binds to exposed hydrophobic sequences in client proteins (residues 385–505); a 10-kDa helical LID domain (residues 506–605); and a 5-kDa dynamically unstructured C-terminal domain (CTD) (residues 606–646) (
Altogether, hsc-70's multiple interactions allow this chaperone to play an important role in several cellular activities, including ribosomal quality control, protein refolding, proteasome-linked degradation, macroautophagy, endosomal microautophagy, chaperone-mediated autophagy, endoplasmic reticulum/Golgi and mitochondrial targeting, and vesicle clathrin uncoating (
Even though it is recognized that membrane surface charges are fundamental for protein targeting, virtually nothing is known about the structural aspects or biophysics of these interactions. Here, we present a study combining biophysical methods (NMR spectroscopy and plasmon resonance) and mutagenesis to delineate the interaction between hsc-70 and PS. The results were validated with endosomal transport assays determining the role of the hsc-70/PS interaction on endosomal microautophagy. We conclude that positively charged residues at the C terminus of the hsc-70 LID domain are interacting most strongly with the negatively charged phosphatidylserine. This interaction is surprisingly specific and involves a few lysine residues. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.
hsc-70 is a molecular chaperone that plays many important roles in proteostasis and protein trafficking. These activities include at least three fundamentally different functions as follows: (i) a role in protein (re)-folding cycles; (ii) a role in guiding proteins to the proteasome; and (iii) a role in guiding proteins into endosomal/lysosomal compartments for macroautophagy, endosomal microautophagy, and chaperone-mediated autophagy (
). hsc-70 carries out each of these activities by interacting with a different set of molecular partners. Although the interaction partners and co-chaperones involved in folding and proteasomal degradation are beginning to be understood (
). Still, virtually nothing is known about the structure or biophysics of these interactions.
Although there were previous reports that hsc-70 interacts with membrane PS, the protein itself does not bear any of the canonical protein lipid binding domains. In this study, we set out to delineate and localize the interaction between hsc-70 and PS with a combination of NMR and mutagenesis studies. We carried out two sets of complementary NMR experiments, hsc-70 interaction with DOPS vesicles and with DOPS nano-discs. As expected for electrostatic interactions, both NMR strategies identified widespread chemical shift perturbations. However, both studies also pointed to larger chemical shift perturbations for the positive residues in the C-terminal tail of the LID. The high density of exposed lysines and arginines in the LID domain appears to be the major site interacting with the negatively charged PS. The C-terminal helix of the LID is not only enriched in positive but also in negative (Glu and Asp) residues (Fig. 5a). In fact, the electrostatic potential for this area of the LID is negative rather than positive (Fig. 5b). However, these negative residues do not show preferential CSPs as do the positive residues (supplemental Figs. S5 and S6). The specificity of interaction is rather surprising. At the outset, one would expect that an electrostatic interaction with a uniformly negatively charged surface of the artificial DOPS vesicles and nano-discs would engage virtually every positively charged residue of the protein.
The lack of broadening of the hsc-70 NMR resonances in the presence of the large DOPS discs (100 Å) or vesicles (1000 Å) indicates mobile binding. Indeed, because NMR line widths are inversely proportional to the mobility of the studied molecules, static binding of hsc-70 SBD (with a rotational correlation time (τc) of 11 ns) to DOPS vesicles (τc of ∼1 μs) would have increased the NMR line width by ∼100-fold rendering the NMR spectrum undetectable. We conclude that hsc-70 bound to the vesicles (and the 100 Å nano-discs) remains mobile, likely by being tethered by the long lysine side chains to a fluid-like surface and possibly by diffusing over the uniform DOPS surface.
Specific and systematic chemical shift perturbations were observed for several resonances when the DOPS nano-discs:hsc-70 stoichiometry was increased from 0 to 1.2 with hsc-70 at 44 μm. The occurrence of shifts at these concentration levels indicates that the affinity of the hsc-70 for the DOPS (nano-discs) must be significantly tighter than 50 μm. DOPS nano-discs are uniform in size and ∼100 Å in diameter. Therefore, they can in principle accommodate a few hsc-70s on each side of the double layer.
Although we may conclude that the DOPS interaction does occur with the C terminus of the hsc-70 LID domain, we cannot fully exclude interactions with other parts of the protein. However, we have tested by NMR the interaction of DOPS with an isolated nucleotide binding domain of hsc-70(1–386) in the ADP state, and we could not discern any changes in the NMR spectrum (results not shown). Furthermore, although we did not test the hsc-70 dynamically unstructured C-terminal tail (residues 610–646), the electrostatic map (Fig. 5, a and b) does not show any positive residues or potential for this range, and therefore no interactions with the PS are expected.
By using plasmon resonance (Fig. 1) we estimate an apparent equilibrium dissociation constant of 4.7 ± 0.1 μm for the interaction of WT/hsc-70 with 100% DOPS vesicles, in 100 mm KCl. This number is precise, but not accurate, because binding of a ligand to a surface with a large (and unknown) number of overlapping binding sites is not completely described by the simple binding equation used to calculate the KD value from the data (
) determined that synthetic peptides, consisting of 3, 5, or 7 lysine residues to vesicles containing 33% negatively charged phospholipid in 100 mm monovalent salt bind with apparent association constants 5 mm, 180 μm, and 7 μm, respectively. They also determined the dependence of the affinity of penta-lysine as a function of negatively charged phospholipid in the composition range 0–50%. Extrapolating their data to 100%, we predict that 3, 5, or 7 lysine residues would bind to 100% DOPS (in 100 mm salt) with apparent association constants of 300 μm, 2 μm, and 7 nm, respectively. Hence, from our experimental KD of 4.7 μm, we estimate that a total of 4–5 lysine residues are involved in the interaction with 100% DOPS vesicles.
Previous studies indicate that hsp-70 interacts differently with different lipids and different modalities under physiological or pathological conditions. During stress-induced cell death, hsp-70 stabilizes lysosomes by binding anionic phospholipids such as bis-(monoacylglycero)phosphate (BMP) (
). The authors concluded that the hsp-70 binding to BMP occurs through both its nucleotide binding domain (W-90) and its substrate binding domain (W-580) and suggested that it relies on tryptophan insertion into the lysosomal membrane. However, under physiological conditions W90 is completely buried, and W580 is partially buried in the hsc-70 or hsp-70 core (versus PDB codes 3HSC and 4PO2) and is unavailable for insertion. It should be noted that hsp-70 binding to BMP is pH-dependent (
In contrast, both hsc-70 and hsp-70 have also been reported to bind other lipids such as PS. This binding did not require insertion into the PS-containing membrane and was mediated by the substrate-binding LID domain (
The NMR measurements and mutagenesis studies presented herein point to the involvement of positively charged residues in the hsc-70 C-terminal LID section for the interaction with negatively charged lipids. As can be seen in Fig. 2, d and h, where the results of the NMR and mutagenesis studies are both visualized on a model of hsc-70 SBD, the correlation is good but not perfect. Several factors can contribute to this partial mismatch. The first and most simple factor is that mutant K569Q was not sufficiently expressed to test the protein for the site with the largest NMR CSP (Fig. 2g). Second, the lysine and arginine NMR CSPs, which report on the chemical environment of the backbone amide groups, likely under-report chemical environmental changes occurring at the peripherally charged groups of these residues. Conversely, an electrostatic field such as that emanating from the DOPS entities may cause shifts on residues that are not truly interacting with those entities. Last and not least, interactions at one site may cause CSPs elsewhere due to conformational changes. Despite all these shortcomings, the fact remains that the NMR CSPs are the largest for some of the lysine side chains that are also implied by mutagenesis.
Other proteins have been shown to use electrostatic interaction surfaces to bind lipid (
). Indeed, this amphiphilic (cationic/hydrophobic) strategy is used by several biological structures to insert into anionic membranes, especially those containing PS. Although PS is present in all cellular membranes, it only confers a negative charge to the plasma and the endosomal membranes; PS present in mitochondria, Golgi, and endoplasmic reticulum is confined to their luminal leaflets and therefore does not charge their cytosolic interfaces (
). It has also been shown that PS directs proteins with strong positive charge to the cytosolic leaflet of the plasma membrane and proteins with moderate positive charge to the cytosolic leaflet of the endosomal membrane (
It is tempting to speculate on the structural origin of the lack of DOPS interaction with hsc-70 in the ATP state (Fig. 1, d and e). Although no structure is known for hsc-70 in this state, it is known for the E. coli homologue DnaK that the LID domain is swept away from the SBD and docked to the NBD (
). Could this be the cause of the ATP-dependent change in DOPS interaction in hsc-70? Regretfully, homology between DnaK and hsc-70 in the LID domain is non-existent beyond residue 584, where we find many interactions with DOPS. This area contains no positively charged residues in DnaK. Hence, the LID docking in the DnaK crystal structures would likely not pertain to hsc-70. Nevertheless, LID docking in hsc-70 could explain the difference in nucleotide-dependent PS binding.
hsc-70 is a hub in cellular proteostasis. The protein interacts with many different molecules, substrates, nucleotides, co-chaperones, and allosteric effectors. Most remarkable is that the protein uses different surfaces for all interactions mapped to date (Fig. 5, c and d). Nucleotides (ATP and ADP) bind into a deep pocket in the NBD (
). The herein delineated endosome interaction surface is at another location again. Our studies reveal a unique surface required for the interaction of hsc-70 with late endosomes that is essential for its role in endosomal microautophagy.
Endosomal microautophagy is an ESCRT-dependent process, which is different from the ESCRT-dependent transport of ubiquitinated plasma membrane proteins into the endosomes, and relies on the cytosolic chaperone hsc-70 for the endosomal internalization of cytosolic proteins (
). Although hsc-70 can bind misfolded and ubiquitinated proteins, we previously demonstrated that in endosomal microautophagy, hsc-70 also binds proteins with KFERQ motifs. Thus, although the endosomal vesiculation, per se, is ESCRT-dependent, cargo internalization into the endosomes involves different chaperones and/or molecular partners when the cargo is membrane-bound or soluble and when it is ubiquitinated or not. The ESCRT-dependent process described in this work is an autophagic process, because it mediates degradation of intracellular proteins inside endo/lysosomes, whereas the ESCRT-mediated degradation of plasma membrane proteins by the endo/lysosomal system has been correctly classified as an endocytic process. We use the term “microautophagy” to differentiate from other cellular forms of autophagy, such as macroautophagy or chaperone-mediated autophagy, and to highlight the morphological similarity of this process to microautophagy described in yeast. However, because the equivalent to the vacuolar yeast in mammals would be lysosomes and this process of autophagy mediated by hsc-70 and ESCRT occurs in endosomes and not in lysosomes, it is necessary to include the term “endosomal” to clarify the compartment where this type of autophagy takes place.
Our work with mutant hsc-70 and isolated endosomes reveals that the positively charged region of the hsc-70-LID is not required for substrate binding to late endosomes but it is absolutely necessary for substrate internalization. Because our structural data and analysis in liposomes reveal that this is the region utilized by hsc-70 to bind PS, it is possible that binding of hsc-70 to PS is the trigger for microvesicle formation and that the latter only forms at membrane micro-domains enriched in PS. Whether hsc-70 binding to PS is directly responsible for the recently described membrane deforming activity of this chaperone (
Importantly, the hsc-70/PS interaction occurring under physiological conditions only requires a 'lateral” interaction with the late endosomal compartments, without protein embedding into the lipid bilayer. Such interaction would facilitate the entry of the hsc-70/cargo into the forming vesicles. In contrast, this interaction is different from what is observed during pathological conditions when lysosomal membrane destabilization requires hsc-70 embedding into the organelle-limiting membrane to preserve endosomal integrity (
Altogether, our data add an additional piece to the puzzle of hsc-70 multivalent interactions. Considering the pivotal role of this protein in cellular proteostasis, there has been considerable interest in generating small molecules and peptides acting as hsc-70 modulators. Our data, providing a novel site for hsc-70 interaction with PS membrane, could provide a novel site for the development of therapeutical hsc-70 modulators.
J. E. G., A. M. C., E. R. P. Z., and L. S. designed the experiments; E. R. P. Z., K. M., C. C. C., S. K., B. S., E. A., A. A., J. N. R., V. C., C. M., and B. S. performed the experiments; K. M., J. E. G., A. M. C., E. R. P. Z., and L. S. wrote the paper.