Molecular mechanism of bacterial Hsp90 pH‐dependent ATPase activity

Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated open‐to‐closed‐to‐open conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pH‐dependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformation‐specific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anti‐correlation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255‐ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255‐ATP salt‐bridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pH‐dependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal site‐specific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity.     

pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation

The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5′-adenylyl-β,γ-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.     

Multiple conformations of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90

Hsp90, an essential eukaryotic chaperone, depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. We used small-angle X-ray scattering and recently developed molecular modeling methods to characterize the solution structure of HtpG and demonstrate how it differs from known Hsp90 conformations. In addition to this HtpG conformation, we demonstrate that under physiologically relevant conditions, multiple conformations coexist in equilibrium. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. These studies provide a unique view of Hsp90 conformational dynamics and provide a model for the role of nucleotide in effecting conformational change.     

From a Ratchet Mechanism to Random Fluctuations Evolution of Hsp90's Mechanochemical Cycle

The 90‐kDa heat shock proteins [heat shock protein 90 (Hsp90)] are a highly conserved ATP-dependent protein family, which can be found from prokaryotic to eukaryotic organisms. In general, Hsp90s are elongated dimers with N- and C-terminal dimerization sites. In a series of publications, we have recently shown that no successive mechanochemical cycle exists for yeast Hsp90 (yHsp90) in the absence of clients or cochaperones. Here, we resolve the mechanochemical cycle of the bacterial homologue HtpG by means of two‐ and three‐color single‐molecule FRET (Förster resonance energy transfer). Unlike yHsp90, the N-terminal dynamics of HtpG is strongly influenced by nucleotide binding and turnover—its reaction cycle is driven by a mechanical ratchet mechanism. However, the C-terminal dimerization site is mainly closed and not influenced by nucleotides. The direct comparison of both proteins shows that the Hsp90 machinery has developed to a more flexible and less nucleotide-controlled system during evolution.     

Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity

Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.     

Ribosomal protein L2 associates with E. coli HtpG and activates its ATPase activity

Although eukaryotic Hsp90 has been studied extensively, the function of its bacterial homologue HtpG remains elusive. Here we report that 50S ribosomal protein L2 was found as an associated protein with His-tagged HtpG from Escherichia coli cultured in minimum medium at 45 °C. L2 specifically activated ATPase activity of HtpG, but other denatured proteins did not. The analysis using domain derivatives of HtpG and L2 showed that C-terminal domain of L2 and the middle to C-terminal domain of HtpG are important for interaction. At physiological salt concentration, L2 was denatured state and was recognized by HtpG as well as other chaperones, DnaK/DnaJ/GrpE and GroEL/GroES. The ATPase of HtpG at increasing concentration of L2 indicated that an L2 molecule bound to a dimer HtpG with apparent K_D of 0.3 μM at 100 mM KCl and 3.3 μM at 200 mM KCl.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin‐proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo‐electron microscopy and X‐ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the β‐subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven α‐subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

Developments,applications, and prospects of cryo‐electron microscopy

Cryo‐electron microscopy (cryo‐EM) is a structural biological method that is used to determine the 3D structures of biomacromolecules. After years of development, cryo‐EM has made great achievements, which has led to a revolution in structural biology. In this article, the principle, characteristics, history, current situation, workflow, and common problems of cryo‐EM are systematically reviewed. In addition, the new development direction of cryo‐EM—cryo‐electron tomography (cryo‐ET), is discussed in detail. Also, cryo‐EM is prospected from the following aspects: the structural analysis of small proteins, the improvement of resolution and efficiency, and the relationship between cryo‐EM and drug development. This review is dedicated to giving readers a comprehensive understanding of the development and application of cryo‐EM, and to bringing them new insights.     

Grp94, the endoplasmic reticulum Hsp90, has a similar solution conformation to cytosolic Hsp90 in the absence of nucleotide

The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide‐dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species‐specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP‐bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X‐ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair‐like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs.     

Osmolyte‐induced conformational changes in the Hsp90 molecular chaperone

Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native‐state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X‐ray scattering we observe dramatic osmolyte‐dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well‐established osmolyte principles that govern stability also apply to large‐scale conformational changes, a proposition that is corroborated by structure‐based fitting of the scattering data, surface area comparisons and m‐value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain‐domain interaction. Hsp90 adopts an additional ligand‐specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.     

Large-Scale Conformational Transitions and Dimerization Are Encoded in the Amino-Acid Sequences of Hsp70 Chaperones

Hsp70s are a class of ubiquitous and highly conserved molecular chaperones playing a central role in the regulation of proteostasis in the cell. Hsp70s assist a myriad of cellular processes by binding unfolded or misfolded substrates during a complex biochemical cycle involving large-scale structural rearrangements. Here we show that an analysis of coevolution at the residue level fully captures the characteristic large-scale conformational transitions of this protein family, and predicts an evolutionary conserved–and thus functional–homo-dimeric arrangement. Furthermore, we highlight that the features encoding the Hsp70 dimer are more conserved in bacterial than in eukaryotic sequences, suggesting that the known Hsp70/Hsp110 hetero-dimer is a eukaryotic specialization built on a pre-existing template.     

HtpG,the prokaryotic homologue of Hsp90, stabilizes a phycobilisome protein in the cyanobacterium Synechococcus elongatus PCC 7942

HtpG, a homologue of HSP90, is essential for thermotolerance in cyanobacteria. It is not known how it plays this important role. We obtained evidence that HtpG interacts with linker polypeptides of phycobilisome in the cyanobacterium Synechococcus elongatus PCC 7942. In an htpG mutant, the 30 kDa rod linker polypeptide was reduced. In vitro studies with purified HtpG and phycobilisome showed that HtpG interacts with the linker polypeptide as well as other linker polypeptides to suppress their thermal aggregation with a stoichiometry of one linker polypeptide/HtpG dimer. We constructed various domain‐truncated derivatives of HtpG to identify putative chaperone sites at which HtpG binds linker polypeptides. The middle domain and the N‐terminal domain, although less efficiently, prevented the aggregation of denatured polypeptides, while the C‐terminal domain did not. Truncation of the C‐terminal domain that is involved in the dimerization of HtpG led to decrease in the anti‐aggregation activity, while fusion of the N‐terminal domain to the middle domain lowered the activity. In vitro studies with HtpG and the isolated 30 kDa rod linker polypeptide provided basically similar results to those with HtpG and phycobilisome. ADP inhibited the anti‐aggregation activity, indicating that a compact ADP conformational state provides weaker aggregation protection compared with the others.     

qFit 3: Protein and ligand multiconformer modeling for X‐ray crystallographic and single‐particle cryo‐EM density maps

New X‐ray crystallography and cryo‐electron microscopy (cryo‐EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open‐source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0 .     

Constitutive Expression of Small Heat Shock Protein in an htpG Disruptant of the Cyanobacterium Synechococcus sp. PCC 7942

In cyanobacteria, a disruptant of hspA encoding a small heat shock protein homologue, shows decreased cell growth rates at moderately high temperatures, and loss of both basal and acquired thermo-tolerances, which resemble the phenotype of an htpG disruptant. In vitro studies have shown that both small heat shock protein and Hsp90 can bind and keep non-native proteins in a refolding-competent state under denaturing conditions. The aim of the present study is to elucidate whether constitutive expression of HspA can functionally replace HtpG, a prokaryotic homolog of Hsp90, in the cyanobacterium Synechococcus sp. PCC 7942. HspA did not improve the viability of the htpG disruptant at a lethal temperature, although it did that of the wild type. It did not improve an iron-starved phenotype of the mutant under normal growth conditions, a novel phenotype found in the present study. These results suggest that cellular function of HtpG may differ significantly from that of HspA.     

HtpG is essential for the thermal stress management in cyanobacteria.

The heat shock protein (Hsp) HtpG is a member of the Hsp90 protein family. We cloned a single-copy gene encoding a homologue of HtpG from the unicellular cyanobacterium Synechococcus sp. PCC 7942. Sequence alignment with HtpGs from other prokaryotes revealed unique features in the cyanobacterial HtpG primary sequence. A monocistronic mRNA of the htpG gene increased transiently in response to heat shock. In order to elucidate the role of HtpG in vivo, we inactivated the htpG gene by targeted mutagenesis. Although the mutation did not affect the photoautotrophic growth at 30 and 42 degrees C, the mutant cells were unable to grow at 45 degrees C. They lost both basal and acquired thermotolerances. These results indicate that HtpG plays an essential role for the thermal stress management in cyanobacteria, the first such an example for either a photosynthetic or a prokaryotic organism.     

Structural basis for HIV‐1 DNA integration in the human genome,role of the LEDGF/P75 cofactor

Integration of the human immunodeficiency virus (HIV‐1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild‐type full‐length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA‐binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral‐induced pathologies.     

Investigating the structural dynamics of the PIEZO1 channel activation and inactivation by coarse‐grained modeling

The PIEZO channels, a family of mechanosensitive channels in vertebrates, feature a fast activation by mechanical stimuli (eg, membrane tension) followed by a slower inactivation. Although a medium‐resolution structure of the trimeric form of PIEZO1 was solved by cryo‐electron microscopy (cryo‐EM), key structural changes responsible for the channel activation and inactivation are still unknown. Toward decrypting the structural mechanism of the PIEZO1 activation and inactivation, we performed systematic coarse‐grained modeling using an elastic network model and related modeling/analysis tools (ie, normal mode analysis, flexibility and hotspot analysis, correlation analysis, and cryo‐EM‐based hybrid modeling and flexible fitting). We identified four key motional modes that may drive the tension‐induced activation and inactivation, with fast and slow relaxation time, respectively. These modes allosterically couple the lateral and vertical motions of the peripheral domains to the opening and closing of the intra‐cellular vestibule, enabling external mechanical forces to trigger, and regulate the activation/inactivation transitions. We also calculated domain‐specific flexibility profiles, and predicted hotspot residues at key domain‐domain interfaces and hinges. Our results offer unprecedented structural and dynamic information, which is consistent with the literature on mutational and functional studies of the PIEZO channels, and will guide future studies of this important family of mechanosensitive channels.