Conformational equilibria in allosteric control of Hsp70 chaperones.

Heat-shock proteins of 70 kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

Targeted photodynamic neutralization of SARS-CoV-2 mediated by singlet oxygen.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has been on a rampage for more than two years. Vaccines in combination with neutralizing antibodies (NAbs) against SARS-CoV-2 carry great hope in the treatment and final elimination of coronavirus disease 2019 (COVID-19). However, the relentless emergence of variants of concern (VOC), including the most recent Omicron variants, presses for novel measures to counter these variants that often show immune evasion. Hereby we developed a targeted photodynamic approach to neutralize SARS-CoV-2 by engineering a genetically encoded photosensitizer (SOPP3) to a diverse list of antibodies targeting the wild-type (WT) spike protein, including human antibodies isolated from a 2003 Severe acute respiratory syndrome (SARS) patient, potent monomeric and multimeric nanobodies targeting receptor-binding domain (RBD), and non-neutralizing antibodies (non-NAbs) targeting the more conserved N-terminal domain (NTD). As confirmed by pseudovirus neutralization assay, this targeted photodynamic approach significantly increased the efficacy of these antibodies, especially that of non-NAbs, against not only the WT but also the Delta strain and the heavily immune escape Omicron strain (BA.1). Subsequent measurement of infrared phosphorescence at 1270 nm confirmed the generation of singlet oxygen (1O2) in the photodynamic process. Mass spectroscopy assay uncovered amino acids in the spike protein targeted by 1O2. Impressively, Y145 and H146 form an oxidization "hotspot", which overlaps with the antigenic "supersite" in NTD. Taken together, our study established a targeted photodynamic approach against the SARS-CoV-2 virus and provided mechanistic insights into the photodynamic modification of protein molecules mediated by 1O2.

Interdomain interactions dictate the function of the Candida albicans Hsp110 protein Msi3.

Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the "holdase" activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain-domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDß interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDß contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the "holdase" activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.

An unexpected second binding site for polypeptide substrates is essential for Hsp70 chaperone activity.

Heat shock proteins of 70 kDa (Hsp70s) are ubiquitous and highly conserved molecular chaperones. They play multiple essential roles in assisting with protein folding and maintaining protein homeostasis. Their chaperone activity has been proposed to require several rounds of binding to and release of polypeptide substrates at the substrate-binding domain (SBD) of Hsp70s. All available structures have revealed a single substrate-binding site in the SBD that binds a single segment of an extended polypeptide of 3-4 residues. However, this well-established single peptide-binding site alone has made it difficult to explain the efficient chaperone activity of Hsp70s. In this study, using purified proteins and site-directed mutagenesis, along with fluorescence polarization and luciferase-refolding assays, we report the unexpected discovery of a second peptide-binding site in Hsp70s. More importantly, the biochemical analyses suggested that this novel binding site, named here P2, is essential for Hsp70 chaperone activity. Furthermore, cross-linking and mutagenesis studies indicated that this second binding site is in the SBD adjacent to the first binding site. Taken together, our results suggest that these two essential binding sites of Hsp70s cooperate in protein folding.

Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec-5 and Siglec-14.

The intracellular chaperone heat-shock protein 70 (Hsp70) can be secreted from cells, but its extracellular role is unclear, as the protein has been reported to both activate and suppress the innate immune response. Potential immunomodulatory receptors on myelomonocytic lineage cells that bind extracellular Hsp70 are not well defined. Siglecs are Ig-superfamily lectins on mammalian leukocytes that recognize sialic acid-bearing glycans and thereby modulate immune responses. Siglec-5 and Siglec-14, expressed on monocytes and neutrophils, share identical ligand-binding domains but have opposing signaling functions. Based on phylogenetic analyses of these receptors, we predicted that endogenous sialic acid-independent ligands should exist. An unbiased screen revealed Hsp70 as a ligand for Siglec-5 and Siglec-14. Hsp70 stimulation through Siglec-5 delivers an anti-inflammatory signal, while stimulation through Siglec-14 is pro-inflammatory. The functional consequences of this interaction are also addressed in relation to a SIGLEC14 polymorphism found in humans. Our results demonstrate that an endogenous non-sialic acid-bearing molecule can be either a danger-associated or self-associated signal through paired Siglecs, and may explain seemingly contradictory prior reports on extracellular Hsp70 action.

Shank3-deficient thalamocortical neurons show HCN channelopathy and alterations in intrinsic electrical properties.

KEY POINTS: Shank3 increases the HCN channel surface expression in heterologous expression systems. Shank3Δ13-16 deficiency causes significant reduction in HCN2 expression and Ih current amplitude in thalamocortical (TC) neurons. Shank3Δ13-16 - but not Shank3Δ4-9 -deficient TC neurons share changes in basic electrical properties which are comparable to those of HCN2-/- TC neurons. HCN channelopathy may critically mediate events downstream from Shank3 deficiency. ABSTRACT: SHANK3 is a scaffolding protein that is highly enriched in excitatory synapses. Mutations in the SHANK3 gene have been linked to neuropsychiatric disorders especially the autism spectrum disorders. SHANK3 deficiency is known to cause impairments in synaptic transmission, but its effects on basic neuronal electrical properties that are more localized to the soma and proximal dendrites remain unclear. Here we confirmed that in heterologous expression systems two different mouse Shank3 isoforms, Shank3A and Shank3C, significantly increase the surface expression of the mouse hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channel. In Shank3Δ13-16 knockout mice, which lack exons 13-16 in the Shank3 gene (both Shank3A and Shank3C are removed) and display a severe behavioural phenotype, the expression of HCN2 is reduced to an undetectable level. The thalamocortical (TC) neurons from the ventrobasal (VB) complex of Shank3Δ13-16 mice demonstrate reduced Ih current amplitude and correspondingly increased input resistance, negatively shifted resting membrane potential, and abnormal spike firing in both tonic and burst modes. Impressively, these changes closely resemble those of HCN2-/- TC neurons but not of the TC neurons from Shank3Δ4-9 mice, which lack exons 4-9 in the Shank3 gene (Shank3C still exists) and demonstrate moderate behavioural phenotypes. Additionally, Shank3 deficiency increases the ratio of excitatory/inhibitory balance in VB neurons but has a limited impact on the electrical properties of connected thalamic reticular (RTN) neurons. These results provide new understanding about the role of HCN channelopathy in mediating detrimental effects downstream from Shank3 deficiency.

A functional DnaK dimer is essential for the efficient interaction with Hsp40 heat shock protein.

Highly conserved molecular chaperone Hsp70 heat shock proteins play a key role in maintaining protein homeostasis (proteostasis). DnaK, a major Hsp70 in Escherichia coli, has been widely used as a paradigm for studying Hsp70s. In the absence of ATP, purified DnaK forms low-ordered oligomer, whereas ATP binding shifts the equilibrium toward the monomer. Recently, we solved the crystal structure of DnaK in complex with ATP. There are two molecules of DnaK-ATP in the asymmetric unit. Interestingly, the interfaces between the two molecules of DnaK are large with good surface complementarity, suggesting functional importance of this crystallographic dimer. Biochemical analyses of DnaK protein supported the formation of dimer in solution. Furthermore, our cross-linking experiment based on the DnaK-ATP structure confirmed that DnaK forms specific dimer in an ATP-dependent manner. To understand the physiological function of the dimer, we mutated five residues on the dimer interface. Four mutations, R56A, T301A, N537A, and D540A, resulted in loss of chaperone activity and compromised the formation of dimer, indicating the functional importance of the dimer. Surprisingly, neither the intrinsic biochemical activities, the ATP-induced allosteric coupling, nor GrpE co-chaperone interaction is affected appreciably in all of the mutations except for R56A. Unexpectedly, the interaction with co-chaperone Hsp40 is significantly compromised. In summary, this study suggests that DnaK forms a transient dimer upon ATP binding, and this dimer is essential for the efficient interaction of DnaK with Hsp40.

Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery.

Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc-based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat-denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co-chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.

Dickkopf-1 drives perineural invasion via PI3K-AKT signaling pathway in head and neck squamous cancer.

Perineural invasion (PNI) leads to the poor prognosis of head and neck squamous cancer (HNSCC) patients, but the mechanism of PNI remains unclear. Dickkopf-1 (DKK1), a secretory protein in the Wnt signaling pathway, was found indeed upregulated in HNSCC cells and tissues. Higher expression of DKK1 was statistically relevant to T stage, N stage, PNI, and poor prognosis of HNSCC. DKK1 overexpression enhanced the migration abilities of cancer cells. Moreover, DKK1-overexpressing cancer cells promoted cancer cells invasion of peripheral nerves in vitro and in vivo. Mechanistically, DKK1 could promote the PI3K-AKT signaling pathway. The migration abilities of neuroblastoma cells, which were enhanced by DKK1-overexpressing HNSCC cell lines, could be reversed by an inhibitor of Akt (MK2206). The association of DKK1 with PNI was also confirmed in HNSCC samples. Variables, including T stage, N stage, DKK1 expression, and PNI, were used to establish a nomogram to predict the survival probability and disease-free probability at 3 and 5 years. In summary, DKK1 can promote the PI3K-AKT signaling pathway in tumor cells and then could induce neuritogenesis and facilitate PNI. MK2206 may be a potential therapeutic target drug for HNSCC patients with PNI.

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity.

The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.

Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data.

Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Zmax ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f'' scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.

Interfacial water molecules contribute to antibody binding to the receptor-binding domain of SARS-CoV-2 spike protein.

Antibodies that recognize the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially the neutralizing antibodies, carry great hope in the treatment and final elimination of COVID-19. Driven by a synchronized global effort, thousands of antibodies against the spike protein have been identified during the past two years, with the structural information available at atomistic detail for hundreds of these antibodies. We developed an improved molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method including explicitly treated interfacial water to calculate the binding free energy between representative antibodies and the receptor binding domain (RBD) domain of SARS-COV-2 spike proteins. We discovered that explicit treatment of water molecules located at the interface between RBD and antibody effectively improves the results for the WT and variants of concern (VOC) systems. Interfacial water molecules, together with surface and internal water molecules, behave drastically from bulk water and exert peculiar impacts on protein dynamics and energy, and thus warrant explicit treatment to complement implicit solvent models. Our results illustrate the importance of including interfacial water molecules to approach efficient and reliable prediction of binding free energy.Communicated by Ramaswamy H. Sarma.

A first-in-class inhibitor of Hsp110 molecular chaperones of pathogenic fungi.

Proteins of the Hsp110 family are molecular chaperones that play important roles in protein homeostasis in eukaryotes. The pathogenic fungus Candida albicans, which causes infections in humans, has a single Hsp110, termed Msi3. Here, we provide proof-of-principle evidence supporting fungal Hsp110s as targets for the development of new antifungal drugs. We identify a pyrazolo[3,4-b] pyridine derivative, termed HLQ2H (or 2H), that inhibits the biochemical and chaperone activities of Msi3, as well as the growth and viability of C. albicans. Moreover, the fungicidal activity of 2H correlates with its inhibition of in vivo protein folding. We propose 2H and related compounds as promising leads for development of new antifungals and as pharmacological tools for the study of the molecular mechanisms and functions of Hsp110s.

Normal-mode-analysis-guided investigation of crucial intersubunit contacts in the cAMP-dependent gating in HCN channels.

Protein structures define a complex network of atomic interactions in three dimensions. Direct visualization of the structure and analysis of the interaction potential energy are not straightforward approaches to pinpoint the atomic contacts that are crucial for protein function. We used the tetrameric hyperpolarization-activated cAMP-regulated (HCN) channel as a model system to study the intersubunit contacts in cAMP-dependent gating. To obtain a systematic survey of the contacts between each pair of residues, we used normal-mode analysis, a computational approach for studying protein dynamics, and constructed the covariance matrix for C-α atoms. The significant contacts revealed by covariance analysis were further investigated by means of mutagenesis and functional assays. Among the mutant channels that show phenotypes different from those of the wild-type, we focused on two mutant channels that express opposite changes in cAMP-dependent gating. Subsequent biochemical assays on isolated C-terminal fragments, including the cAMP binding domain, revealed only minimal effects on cAMP binding, suggesting the necessity of interpreting the cAMP-dependent allosteric regulation at the whole-channel level. For this purpose, we applied the patch-clamp fluorometry technique and observed correlated changes in the dynamic, state-dependent cAMP binding in the mutant channels. This study not only provides further understanding of the intersubunit contacts in allosteric coupling in the HCN channel, it also illustrates an effective strategy for delineating important atomic contacts within a structure.

A novel and unique ATP hydrolysis to AMP by a human Hsp70 Binding immunoglobin protein (BiP).

Hsp70s are ubiquitous and highly conserved molecular chaperones. They play crucial roles in maintaining cellular protein homeostasis. It is well established that Hsp70s use the energy of ATP hydrolysis to ADP to power the chaperone activity regardless of the cellular locations and isoforms. Binding immunoglobin protein (BiP), the major member of Hsp70s in the endoplasmic reticulum, is essential for protein folding and quality control. Unexpectedly, our structural analysis of BiP demonstrated a novel ATP hydrolysis to AMP during crystallization under the acidic conditions. Our biochemical studies confirmed this newly discovered ATP to AMP hydrolysis in solutions. Unlike the canonical ATP to ADP hydrolysis observed for Hsp70s, this ATP hydrolysis to AMP depends on the substrate-binding domain of BiP and is inhibited by the binding of a peptide substrate. Intriguingly, this ATP to AMP hydrolysis is unique to BiP, not shared by two representative Hsp70 proteins from the cytosol. Taken together, this novel and unique ATP to AMP hydrolysis may provide a potentially new direction for understanding the activity and cellular function of BiP.

State-dependent cAMP binding to functioning HCN channels studied by patch-clamp fluorometry.

One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed a robust, close-to-threefold increase in ligand binding, which was more pronounced at subsaturating ligand concentrations than higher concentrations. This observation supported the cyclic allosteric models and indicated that protein allostery can be implemented through differentiating ligand binding affinities between resting and active states. The kinetics of ligand binding largely matched channel activation. However, during channel deactivation, ligand unbinding was slower than channel closing, suggesting a delayed response to membrane potential by the ligand binding machinery. Our results provide what we believe to be new insights into the cAMP-dependent gating in HCN channel and the interpretation of protein allostery for general ligand-gated channels and receptors.

Structural basis for the cAMP-dependent gating in the human HCN4 channel.

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between ß4 and ß5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.

Purification and biochemical characterization of Msi3, an essential Hsp110 molecular chaperone in Candida albicans.

Hsp110s are unique and essential molecular chaperones in the eukaryotic cytosol. They play important roles in maintaining cellular protein homeostasis. Candida albicans is the most prevalent yeast opportunistic pathogen that causes fungal infections in humans. As the only Hsp110 in Candida albicans, Msi3 is essential for the growth and infection of Candida albicans. In this study, we have expressed and purified Msi3 in nucleotide-free state and carried out biochemical analyses. Sse1 is the major Hsp110 in budding yeast S. cerevisiae and the best characterized Hsp110. Msi3 can substitute Sse1 in complementing the temperature-sensitive phenotype of S. cerevisiae carrying a deletion of SSE1 gene although Msi3 shares only 63.4% sequence identity with Sse1. Consistent with this functional similarity, the purified Msi3 protein shares many similar biochemical activities with Sse1 including binding ATP with high affinity, changing conformation upon ATP binding, stimulating the nucleotide-exchange for Hsp70, preventing protein aggregation, and assisting Hsp70 in refolding denatured luciferase. These biochemical characterizations suggested that Msi3 can be used as a model for studying the molecular mechanisms of Hsp110s.