Substrate-binding Domain Research Articles

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b’ domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b’ domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.

Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.

NLRP6 suppresses gastric cancer growth via GRP78 ubiquitination

Nod-like receptor pyrin domain-containing protein 6 (NLRP6) plays a key role in innate immunity, host defense and tumorigenesis. Our previous study has demonstrated the tumor suppressor role of NLRP6 in gastric cancer. In the present study, we explored the interaction protein of NLRP6 by Flag-tagged immunoprecipitation assay and liquid chromatography/mass spectrometry-based proteomics analysis. The 78 kDa glucose-regulated protein (GRP78), a heat shock protein, was identified as an interaction protein of NLRP6. The binding of NLRP6 to GRP78 was through the Pyrin domain, and the substrate binding domain (SBD) domain of GRP78 was responsible for the interaction with NLRP6. NLRP6 overexpression enhanced the polyubiquitination of GRP78 in gastric cancer cells. Overexpression of GRP78 abolished the effects of NLRP6 overexpression in gastric cancer cell proliferation, cell cycle progression, cell apoptosis, migration and Cyclin D1 expression. GRP78 knockdown reversed the effects of NLRP6 knockdown on cell proliferation and cell cycle progression. NLRP6 expression was negatively correlated with GRP78 expression in human gastric tissues. Tumorigenicity assay indicated that GRP78 mediated the functions of NLRP6 on gastric cancer cell growth in vivo. ON-013100, which could inhibit Cyclin D1 expression, was less effective in treating xenografts of gastric cancer cells with higher level of NLRP6 than in those with lower level of NLRP6. In conclusion, our study suggested that NLRP6 exerted inhibitory effects on gastric cancer cell growth by promoting the ubiquitination of GRP78.

Preliminary Virtual Screening Studies to Identify GRP78 Inhibitors Which May Interfere with SARS-CoV-2 Infection.

SARS-CoV-2 Spike protein was predicted by molecular docking to bind the host cell surface GRP78, which was suggested as a putative good molecular target to inhibit Covid-19. We aimed to confirm that GRP78 gene expression was increased in blood of SARS-CoV-2 (+) versus SARS-CoV-2 (−) pneumonia patients. In addition, we aimed to identify drugs that could be repurposed to inhibit GRP78, thus with potential anti-SARS-CoV-2 activity. Gene expression studies were performed in 10 SARS-CoV-2 (−) and 24 SARS-CoV-2 (+) pneumonia patients. A structure-based virtual screen was performed with 10,761 small molecules retrieved from DrugBank, using the GRP78 nucleotide binding domain and substrate binding domain as molecular targets. Results indicated that GRP78 mRNA levels were approximately four times higher in the blood of SARS-CoV-2 (+) versus SARS-CoV-2 (−) pneumonia patients, further suggesting that GRP78 might be a good molecular target to treat Covid-19. In addition, a total of 409 compounds were identified with potential as GRP78 inhibitors. In conclusion, we found preliminary evidence that further proposes GRP78 as a possible molecular target to treat Covid-19 and that many clinically approved drugs bind GRP78 as an off-target effect. We suggest that further work should be urgently carried out to confirm if GRP78 is indeed a good molecular target and if some of those drugs have potential to be repurposed for SARS-CoV-2 antiviral activity.

Zika virus envelope – heat shock protein A5 (GRP78) binding site prediction

Recent studies reported the association of the Zika virus (ZIKV) with a stress response receptor on the host cell membrane that facilitates viral entry. This host receptor was the heat shock protein A5 (HSPA5), also termed glucose-regulating protein 78 (GRP78). In this study, structural bioinformatics and molecular dynamics simulations were utilized to suggest the binding site of ZIKV envelope protein during the interaction with cell-surface GRP78. The Pep42 cyclic peptide was used as a profiler, as it was reported earlier, to target GRP78 on the cancer cell membrane selectively. Sequence and structural alignments show that part of the ZIKV envelope protein (C308-C339 region), in addition to its cyclic nature, has somehow sequence and structural similarities to the cyclic Pep42. Three amino acids in the ZIKV envelope were identical to those in the Pep42 peptide. Cyclic peptides dynamics are studied, and its binding to GRP78 is predicted. Protein-protein docking is further performed to explore the binding characteristics of the ZIKV envelope to GRP78. Results revealed that the binding was favorable between ZIKV envelope protein and GRP78. The docking pose revealed the involvement of the substrate-binding domain ß of GRP78 and the domain III of the ZIKV envelope protein in viral recognition for the host-cell.

Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone

Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

Host cell factors stimulate HIV-1 transcription by antagonizing substrate-binding function of Siah1 ubiquitin ligase to stabilize transcription elongation factor ELL2.

The Siah1 and Siah2 ubiquitin ligases are implicated in diverse biological processes ranging from cellular stress responses, signaling to transcriptional regulation. A key substrate of Siah1 is ELL2, which undergoes proteolysis upon polyubiquitination. ELL2 stimulates transcriptional elongation and is a subunit of the Super Elongation Complex (SEC) essential for HIV-1 transactivation. Previously, multiple transcriptional and post-translational mechanisms are reported to control Siah's expression and activity. Here we show that the activity of Siah1/2 can also be suppressed by host cell factor 1 (HCF1), and the hitherto poorly characterized HCF2, which themselves are not degraded but can bind and block the substrate-binding domain (SBD) of Siah1/2 to prevent their autoubiquitination and trans-ubiquitination of downstream targets including ELL2. This effect stabilizes ELL2 and enhances the ELL2-SEC formation for robust HIV-1 transactivation. Thus, our study not only identifies HCF1/2 as novel activators of HIV-1 transcription through inhibiting Siah1 to stabilize ELL2, but also reveals the SBD of Siah1/2 as a previously unrecognized new target for HCF1/2 to exert this inhibition.

Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.

Growth-Regulated Hsp70 Phosphorylation Regulates Stress Responses and Prion Maintenance.

Maintenance of protein homeostasis in eukaryotes under normal growth and stress conditions requires the functions of Hsp70 chaperones and associated cochaperones. Here, we investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. Ser151 phosphorylation in yeast Hsp70 (Ssa1) is promoted by cyclin-dependent kinase (Cdk1) during normal growth. Phosphomimetic substitutions at this site (S151D) dramatically downregulate heat shock responses, a result conserved with HSC70 S153 in human cells. Phosphomimetic forms of Ssa1 also fail to relocalize in response to starvation conditions, do not associate in vivo with Hsp40 cochaperones Ydj1 and Sis1, and do not catalyze refolding of denatured proteins in vitro in cooperation with Ydj1 and Hsp104. Despite these negative effects on HSC70/HSP70 function, the S151D phosphomimetic allele promotes survival of heavy metal exposure and suppresses the Sup35-dependent [PSI+ ] prion phenotype, consistent with proposed roles for Ssa1 and Hsp104 in generating self-nucleating seeds of misfolded proteins. Taken together, these results suggest that Cdk1 can downregulate Hsp70 function through phosphorylation of this site, with potential costs to overall chaperone efficiency but also advantages with respect to reduction of metal-induced and prion-dependent protein aggregate production.

Article of Significant Interest in This Issue

Maintenance of protein homeostasis requires the functions of Hsp70 chaperones. Kao et al. (e00628-19) investigate an evolutionarily conserved serine phosphorylation that occurs at the site of communication between the nucleotide-binding and substrate-binding domains of Hsp70. They found that phosphomimetic substitutions at this site dramatically downregulated heat shock responses and relocalization in response to starvation in budding yeast and in mammalian cells and blocked refolding of denatured proteins in vitro. Despite these negative effects, the phosphomimetic allele promoted survival of heavy metals and suppressed the [PSI+] prion phenotype in yeast, consistent with proposed roles for Hsp70 in generating self-nucleating seeds of misfolded proteins.

Identification and characterization of a novel extracellular polyhydroxyalkanoate depolymerase in the complete genome sequence of Undibacterium sp. KW1 and YM2 strains.

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that is synthesized by a wide range of microorganisms. One of the derivatives of PHA, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) has flexible material properties and low melting temperature. We have previously demonstrated that PHBH is degradable in a freshwater environment via the formation of biofilm on the surface of the PHBH film. Undibacterium sp. KW1 and YM2, which were isolated from the biofilm present on the PHBH film in the freshwater sample, were shown to have PHBH-degrading activity. In this study, the complete genome sequence of KW1 and YM2 revealed that the extracellular PHA depolymerase gene, designated as phaZUD, was present in their chromosomes. Sequence analysis revealed that PhaZUD contained four domains: a signal peptide, catalytic domain, linker domain, and substrate-binding domain. Escherichia coli harboring a PhaZUD-expressing plasmid showed PHBH-degrading activity in LB medium containing 1 wt% PHBH powder. The recombinant His-tagged PhaZUD from KW1 and YM2 was purified from the culture supernatant and shown to have PHBH-degrading activity at the optimum temperature of 35 and 40°C, respectively. When the degradation product in the PHBH solution was treated with PhaZUD and assayed by LC-TOF-MS, we detected various oligomer structures, but no more than pentamers, which consist of 3-hydroxybutyrate and 3-hydroxyhexanoate. These results demonstrated that PhaZUD may have an endo-type extracellular PHA depolymerase activity.

Natural products may interfere with SARS-CoV-2 attachment to the host cell

SARS-CoV-2 has been emerged in December 2019 in China, causing deadly (5% mortality) pandemic pneumonia, termed COVID-19. More than one host-cell receptor is reported to be recognized by the viral spike protein, among them is the cell-surface Heat Shock Protein A5 (HSPA5), also termed GRP78 or BiP. Upon viral infection, HSPA5 is upregulated, then translocating to the cell membrane where it is subjected to be recognized by the SARS-CoV-2 spike. In this study, some natural product compounds are tested against the HSPA5 substrate-binding domain β (SBDβ), which reported to be the recognition site for the SARS-CoV-2 spike. Molecular docking and molecular dynamics simulations are used to test some natural compounds binding to HSPA5 SBDβ. The results show high to a moderate binding affinity for the phytoestrogens (Diadiazin, Genistein, Formontein, and Biochanin A), chlorogenic acid, linolenic acid, palmitic acid, caffeic acid, caffeic acid phenethyl ester, hydroxytyrosol, cis-p-Coumaric acid, cinnamaldehyde, thymoquinone, and some physiological hormones such as estrogens, progesterone, testosterone, and cholesterol to the HSPA5 SBDβ. Based on its binding affinities, the phytoestrogens and estrogens are the best in binding HSPA5, hence may interfere with SARS-CoV-2 attachment to the stressed cells. These compounds can be successful as anti-COVID-19 agents for people with a high risk of cell stress like elders, cancer patients, and front-line medical staff.Communicated by Ramaswamy H. Sarma

Functional diversity between HSP70 paralogs caused by variable interactions with specific co-chaperones

Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis-associated protein variant superoxide dismutase 1 (SOD1)-A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

KLR-70: A Novel Cationic Inhibitor of the Bacterial Hsp70 Chaperone.

The heat-shock factor Hsp70 and other molecular chaperones play a central role in nascent protein folding. Elucidating the task performed by individual chaperones within the complex cellular milieu, however, has been challenging. One strategy for addressing this goal has been to monitor protein biogenesis in the absence and presence of inhibitors of a specific chaperone, followed by analysis of folding outcomes under both conditions. In this way, the role of the chaperone of interest can be discerned. However, development of chaperone inhibitors, including well-known proline-rich antimicrobial peptides, has been fraught with undesirable side effects, including decreased protein expression yields. Here, we introduce KLR-70, a rationally designed cationic inhibitor of the Escherichia coli Hsp70 chaperone (also known as DnaK). KLR-70 is a 14-amino acid peptide bearing naturally occurring residues and engineered to interact with the DnaK substrate-binding domain. The interaction of KLR-70 with DnaK is enantioselective and is characterized by high affinity in a buffered solution. Importantly, KLR-70 does not significantly interact with the DnaJ and GroEL/ES chaperones, and it does not alter nascent protein biosynthesis yields across a wide concentration range. Some attenuation of the anti-DnaK activity of KLR-70, however, has been observed in the complex E. coli cell-free environment. Interestingly, the d enantiomer D-KLR-70, unlike its all-L KLR-70 counterpart, does not bind the DnaK and DnaJ chaperones, yet it strongly inhibits translation. This outcome suggests that the two enantiomers (KLR-70 and D-KLR-70) may serve as orthogonal inhibitors of chaperone binding and translation. In summary, KLR-70 is a novel chaperone inhibitor with high affinity and selectivity for bacterial Hsp70 and with considerable potential to help in parsing out the role of Hsp70 in nascent protein folding.

Global Regulatory Roles of the Histidine-Responsive Transcriptional Repressor HutC in Pseudomonas fluorescens SBW25.

HutC is known as a transcriptional repressor specific for histidine utilization (hut) genes in Gram-negative bacteria, including Pseudomonas fluorescens SBW25. However, its precise mode of protein-DNA interactions hasn't been examined with purified HutC proteins. Here, we performed electrophoretic mobility shift assay (EMSA) and DNase I footprinting using His6-tagged HutC and biotin-labeled probe of the hut promoter (PhutU). Results revealed a complex pattern of HutC oligomerization, and the specific protein-DNA interaction is disrupted by urocanate, a histidine derivative, in a concentration-dependent manner. Next, we searched for putative HutC-binding sites in the SBW25 genome. This led to the identification of 143 candidate targets with a P value less than 10-4 HutC interaction with eight selected candidate sites was subsequently confirmed by EMSA analysis, including the type IV pilus assembly protein PilZ, phospholipase C (PlcC) for phosphatidylcholine hydrolyzation, and key regulators of cellular nitrogen metabolism (NtrBC and GlnE). Finally, an isogenic hutC deletion mutant was subjected to transcriptome sequencing (RNA-seq) analysis and phenotypic characterization. When bacteria were grown on succinate and histidine, hutC deletion caused upregulation of 794 genes and downregulation of 525 genes at a P value of <0.05 with a fold change cutoff of 2.0. The hutC mutant displayed an enhanced spreading motility and pyoverdine production in laboratory media, in addition to the previously reported growth defect on the surfaces of plants. Together, our data indicate that HutC plays global regulatory roles beyond histidine catabolism through low-affinity binding with operator sites located outside the hut locus.IMPORTANCE HutC in Pseudomonas is a representative member of the GntR/HutC family of transcriptional regulators, which possess a N-terminal winged helix-turn-helix (wHTH) DNA-binding domain and a C-terminal substrate-binding domain. HutC is generally known to repress expression of histidine utilization (hut) genes through binding to the PhutU promoter with urocanate (the first intermediate of the histidine degradation pathway) as the direct inducer. Here, we first describe the detailed molecular interactions between HutC and its PhutU target site in a plant growth-promoting bacterium, P. fluorescens SBW25, and further show that HutC possesses specific DNA-binding activities with many targets in the SBW25 genome. Subsequent RNA-seq analysis and phenotypic assays revealed an unexpected global regulatory role of HutC for successful bacterial colonization in planta.

Biophysical Folding Sensors for the Detection of Misfolded Proteins by Hsp70

Chaperoned ubiquitination complexes play a defining role in responding to chemical and pathological stresses and an understanding of how these complexes are regulated on a molecular level offers the opportunity to identify new avenues of treatment for a wide range of diseases. Hsp70 participates in chaperoned ubiquitination by recruiting and attempting to refold misfolded protein “clients” in an ATP‐dependent process. Misfolded clients interact with a binding cleft within the substrate binding domain (SBD) formed by a beta‐sheet subdomain (SBD‐beta) and helical lid subdomain. Understanding how Hsp70 recruits clients and positions clients relative to the E3 ligase CHIP is critical for understanding the core interactions between CHIP and Hsp70 that dictate the outcome of chaperoned ubiquitination. Herein we describe a set of folding sensors representing a spectrum of folded and unfolded states. When utilized in ubiquitination reactions this system provides a tunable folded/unfolded client with an Hsp70‐SBD binding site that provides control over the distance from the model unfolded client to the E2~Ub active site bound to the E3 ligase CHIP, thereby providing a measure of how folding state dictates degree of ubiquitination.Support or Funding InformationNIGMS R35 GM128595

Exploring Hsp70 chaperone interactions with a full‐length substrate

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding and preventing proteins from aggregation (Clérico et al., 2015). Despite extensive research that reveals a selective promiscuity between the Hsp70 substrate‐binding domain (SBD) and client proteins, there is a paucity of data on the interaction of Hsp70s with large protein clients. To investigate the interaction between Hsp70s and a full‐length substrate, we chose to characterize the binding of the E. coli homolog,DnaK, to the precursor of alkaline phosphatase (proPhoA). proPhoA is an endogenous substrate of DnaK that is unfolded and soluble under reducing conditions, and contains at least five sequences that bind DnaK when presented as short peptides (Rüdiger et al., 1997; Wild et al., 1992). To simplify the experimental conditions we used truncated versions of proPhoA (~100 residues long) that contain 1 or 2 binding sites. We used biophysical techniques to reveal the affinity of binding, and NMR spectroscopy to elucidate the mode of chaperone binding to the proPhoA fragments. We found that there is a hierarchy of affinities among the 5 potential binding sites, and novel cryptic binding sites have been observed. In addition, data suggest there may be a role for residues beyond the canonical five‐residue core sequences. The mechanism of DnaK binding site selection on a substrate containing multiple potential sites and the structural basis of interaction between the chaperone and its respective binding sites on a full‐length substrate will be described.Support or Funding InformationNIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/2021

Functional Regulation of Hsp70 Allostery by Post‐Translational Modifications (PTMs)

The 70‐kDa heat shock proteins (Hsp70s) perform critical functions in the protein homeostasis network, such as assisting nascent chain folding, preventing protein misfolding and aggregation, facilitating assembly of complexes, and driving protein translocation. The function of Hsp70s relies on a conserved allosteric mechanism involving substrate binding and release modulated by ATP binding and hydrolysis, with the participation of co‐chaperones, including J‐proteins and nucleotide exchange factors. Many studies in recent years have demonstrated that post‐translational modifications (PTMs) play important roles in functional regulations of Hsp70s. As an example, a recent report showed that phosphorylation of Hsp70 T495 upon infection by Legionella pneumophila inhibits host protein translation. While the modulation of Hsp70 functions can be achieved by a variety of means, including tuning its allosteric landscape, altering its substrate binding properties, varying its co‐chaperone interaction, etc., the mechanism of functional regulation of Hsp70s through PTMs is still unknown. In the current study, by using nuclear magnetic resonance spectroscopy (NMR) and biophysical/biochemical assays based on phosphorylation‐mimicking mutant, we are able to elucidate the origin of functional regulation by T495 phosphorylation in Hsp70. Phosphorylation of T495 shifts Hsp70 to the conformation favored by ATP binding in which the nucleotide‐binding domain (NBD) and substrate‐binding domain (SBD) are intimately docked, thus reducing the substrate binding activity of the molecular chaperone. These results underline the regulation of Hsp70 function by modulating its allosteric landscape through PTMs.Support or Funding InformationSupported by NIH grant GM118161