N-terminal Nucleotide-binding Domain Research Articles

The analysis of inducible family members in the water flea Daphnia magna led to the identification of an uncharacterized lineage of heat shock protein 70

To explore the function and evolutionary relationships of inducible heat shock protein 70 (Hsp70) in Daphnia magna, cDNAs of four Hsp70 family members (DmaHsp70, DmaHsp70-2, DmaHsp70-12, DmaHsp70-14) were cloned. While all DmaHsp70s possess three function domains, it is noteworthy that only DmaHsp70 ends with a “EEVD” motif. Phylogenetic analysis indicates that the Hsp70-12 lineage is distanced from the rest, and therefore it is an uncharacterized lineage of Hsp70. The differences in isoelectric point and 3-dimensional (3D) conformation of the N-terminal nucleotide binding domain (NBD) of DmaHsp70s further support the theory. DmaHsp70s exhibit varied motif distribution patterns and the logo sequences of motifs have diverse signature characteristics, indicating that different mechanisms are involved in the regulation of ATP binding and hydrolysis for the DmaHsp70s. Protein-protein network together with the predicted subcellular locations of DmaHsp70s suggest that they likely fulfill distinct roles in cells. The transcription of four DmaHsp70s were changed during the recovery stage after thermal stress or oxidative stress. But the expression pattern of them were dissimilar. Collectively, these results collectively elucidated the identification of a previously uncharacterizedHsp70 lineage in animal and extended our understanding of the Hsp70 family.

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.

Genome-wide identification, phylogenetic relationships and expression patterns of the NOD-like receptor (NLR) gene family in flounder (Paralichthys olivaceus)

NOD-like receptors (NLRs) are one of the pattern recognition receptors which have been widely known for identifying pathogens and regulating innate immunity in mammals, but the functions of the NLR gene family in teleost fish remain poorly understood. In this study, we conducted a comprehensive identification and analysis of the flounder (Paralichthys olivaceus) NLR gene family, including bioinformatics information, evolutionary relationships, gene structures, conserved motifs, domain composition, expression patterns and protein-protein interaction (PPI). We identified 22 NLRs in flounder (flNLRs) which were clustered into three subfamilies according to their domain organizations and phylogenetic features, i.e., NLR-A (6 members) resembling mammalian NODs, NLR-B (1 member) resembling mammalian NLRPs, and NLR-C (15 members) unique to teleost fish. All flNLRs shared a conserved NACHT domain including an N-terminal nucleotide-binding domain, a middle helical domain 1, and a winged helix domain. Gene structure analysis displayed that flNLRs were significantly different, with exon numbers from 1 to 52. Conserved domain analysis showed that the N-terminus of flNLRs possessed different characteristics of the domains including CARD domain, PYRIN domain, RING domain, and fish-specific FISNA domain, and the C-terminus of seven NLR-C members contained an extra B30.2 domain, named NLRC-B30.2 group. Notably, flNLRs were expressed in all nine tested tissues, showing higher expressions in the systemic and mucosal immune tissues (e.g., kidney, spleen, hindgut, gills, skin, liver) in healthy flounder, and significant responses to intraperitoneal injection and immersion immunization of inactivated Vibrio anguillarum in mucosal tissues, especially the NLR-C members. In addition, PPI analysis demonstrated that some flNLRs of NLR-A and NLR-C shared the same interacting proteins such as RIPK2, TRAF6, MAVS, CASP, ASC, and ATG5, suggesting they might play crucial roles in host defense, antiviral innate immunity, inflammation, apoptosis and autophagy. This study for the first time characterized the NLR gene family of flounder at the genome-wide level, and the results provided a better understanding of the evolution of the NLR gene family and their immune functions in innate immunity in fish.

Direct observation of chemo-mechanical coupling in DnaK by single-molecule force experiments

Protein allostery requires a communication channel for functional regulation between distal sites within a protein. In the molecular chaperone Hsp70, a two-domain enzyme, the ATP/ADP status of an N-terminal nucleotide-binding domain regulates the substrate affinity of a C-terminal substrate-binding domain. Recently available three-dimensional structures of Hsp70 in ATP/ADP states have provided deep insights into molecular pathways of allosteric signals. However, direct mechanical probing of long-range allosteric coupling between the ATP hydrolysis step and domain states is missing. Using laser optical tweezers, we examined the mechanical properties of a truncated two-domain DnaK(1–552ye) in apo/ADP/ATP- and peptide-bound states. We find that in the apo and ADP states, DnaK domains are mechanically stable and rigid. However, in the ATP state, substrate-binding domain (SBD)∗ye is mechanically destabilized as the result of interdomain docking followed by the unfolding of the α-helical lid. By observing the folding state of the SBD, we could observe the continuous ATP/ADP cycling of the enzyme in real time with a single molecule. The SBD lid closure is strictly coupled to the chemical steps of the ATP hydrolysis cycle even in the presence of peptide substrate.

Neutron crystallographic analysis of the nucleotide-binding domain of Hsp72 in complex with ADP

The 70 kDa heat-shock proteins (Hsp70s) are ATP-dependent molecular chaperones that contain an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain. Hsp70s bind to misfolded/unfolded proteins and thereby prevent their aggregation. The ATP hydrolysis reaction in the NBD plays a key role in allosteric control of the binding of substrate proteins. In the present study, the neutron crystal structure of the NBD of Hsp72, a heat-inducible Hsp70 family member, was solved in complex with ADP in order to study the structure–function relationship with a focus on hydrogens. ADP bound to Hsp72 was fully deprotonated, and the catalytically important residues, including Asp10, Asp199 and Asp206, are also deprotonated. Neutron analysis also enabled the characterization of the water clusters in the NBD. Enzymatic assays and X-ray crystallographic analysis revealed that the Y149A mutation exhibited a higher ATPase activity and caused disruption of the water cluster and incorporation of an additional magnesium ion. Tyr149 was suggested to contribute to the low intrinsic ATPase activity and to stabilize the water cluster. Collectively, these structural studies will help to elucidate the molecular basis of the function of Hsp72.

HSPA6 and its role in cancers and other diseases.

Heat Shock Protein Family A (Hsp70) Member 6 (HSPA6) (Online Mendelian Inheritance in Man: 140555) belongs to the HSP70 family and is a partially conserved inducible protein in mammals. The HSPA6 gene locates on the human chromosome 1q23.3 and encodes a protein containing two important structural domains: The N-terminal nucleotide-binding domain and the C-terminal substrate-binding domain. Currently, studies have found that HSPA6 not only plays a role in the tumorigenesis and tumor progresses but also causes non-tumor-related diseases. Furthermore, HSPA6 exhibits to inhibit tumorigenesis and tumor progression in some types of cancers but promotes in others. Even though HSPA6 research has increased, its exact roles and mechanisms are still unclear. This article reviews the structure, expression, function, research progress, possible mechanism, and perspective of HSPA6 in cancers and other diseases, highlighting its potential role as a targeted therapeutic and prognostic marker.

Hsp90 Inhibitor STA9090 Sensitizes Hepatocellular Carcinoma to Hyperthermia-Induced DNA Damage by Suppressing DNA-PKcs Protein Stability and mRNA Transcription.

As a conserved molecular chaperone, heat shock protein 90 (Hsp90) maintains the stability and homeostasis of oncoproteins and helps cancer cells survive. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a pivotal role in the non-homologous end joining pathway for DNA double-strand breaks (DSB) repair. Tumor cells contain higher levels of DNA-PKcs to survive by the hostile tumor microenvironment and various antitumor therapies. Here, we showed that increased levels of Hsp90α, Hsp90β, and DNA-PKcs correlated with a poor overall survival in hepatocellular carcinoma (HCC). We revealed that Hsp90 N-terminal domain and C-terminal domain have different effects on DNA-PKcs protein and mRNA levels. The stability of DNA-PKcs depended on Hsp90α N-terminal nucleotide binding domain. Transcription factor SP1 regulates the transcription of PRKDC (gene name of DNA-PKcs) and is a client protein of Hsp90. Inhibition of Hsp90 N-terminal by STA9090 decreased the location of Hsp90α in nucleus, Hsp90α-SP1 interaction, SP1 level, and the binding of Hsp90α/SP1 at the proximal promoter region of PRKDC Because hyperthermia induces DSBs with increases level of DNA-PKcs, combined STA9090 treatment with hyperthermia effectively delayed the tumor growth and significantly decreased DNA-PKcs levels in xenografts model. Consistently, inhibition of Hsp90 increased the number of heat shock-induced γ-H2AX foci and delayed the repair of DSBs. Altogether, our results suggest that Hsp90 inhibitor STA9090 decreases DNA-PKcs protein stability and PRKDC mRNA level, which provide a theoretical basis for the promising combination therapy of hyperthermia and Hsp90 inhibitor in HCC.

Intermediates in allosteric equilibria of DnaK–ATP interactions with substrate peptides

Hsp70 molecular chaperones facilitate protein disaggregation and proper folding through iterative cycles of polypeptide binding and release that are allosterically coupled to ATP binding and hydrolysis. Hsp70s are ubiquitous and highly conserved across all of life; they bind ATP at an N-terminal nucleotide-binding domain (NBD) and client peptides in the substrate-binding domain (SBD). The NBD and SBD are connected by a highly conserved linker segment that is integrated into the NBD when ATP is bound but is flexible when the NBD is nucleotide-free or bound with ADP. Allosteric coupling is lost when the linker is flexible, and the freed SBD binds peptide clients with high affinity. It was recently discovered that Hsp70-ATP is in an equilibrium between a restraining state (R) with little affinity for peptides and a low ATPase activity, and a stimulating state (S) that binds peptides efficiently, but with rapid kinetics, and has a relatively high ATPase activity. While attempting to characterize the S state, crystal structures of DnaK-ATP were obtained that demonstrate intrinsic Hsp70 plasticity that affects binding interactions with substrate peptides. These structures provide insights into intermediate states along transition pathways in the Hsp70 chaperone cycle.

Discovery of novel Hsp90 C-terminal domain inhibitors that disrupt co-chaperone binding

Heat shock protein 90 (Hsp90) is an essential molecular chaperone that performs vital stress-related and housekeeping functions in cells and is a current therapeutic target for diseases such as cancers. Particularly, the development of Hsp90 C-terminal domain (CTD) inhibitors is highly desirable as inhibitors that target the N-terminal nucleotide-binding domain may cause unwanted biological effects. Herein, we report on the discovery of two drug-like novel Hsp90 CTD inhibitors by using virtual screening and intrinsic protein fluorescence quenching binding assays, paving the way for future development of new therapies that employ molecular chaperone inhibitors.

MAPKAP kinase 2-mediated phosphorylation of HspA1L protects male germ cells from heat stress-induced apoptosis.

Developing male germ cells are extremely sensitive to heat stress; consequently, anatomic and physiologic adaptations have evolved to maintain proper thermoregulation during mammalian spermatogenesis. At the cellular level, increased expression and activity of HSP70 family members occur in response to heat stress in order to refold partially denatured proteins and restore function. In addition, several kinase-mediated signaling pathways are activated in the testis upon hyperthermia. The p38 MAP kinase (MAPK) pathway plays an important role in mitigating heat stress, and recent findings have implicated the downstream p38 substrate, MAPKAP kinase 2 (MK2), in this process. However, the precise function that this kinase plays in spermatogenesis is not completely understood. Using a proteomics-based screen, we identified and subsequently validated that the testis-enriched HSP70 family member, HspA1L, is a novel substrate of MK2. We demonstrate that MK2 phosphorylates HspA1L solely on Ser241, a residue within the N-terminal nucleotide-binding domain of the enzyme. This phosphorylation event enhances the chaperone activity of HspA1L in vitro and renders male germ cells more resistant to heat stress-induced apoptosis. Taken together, these findings illustrate a novel stress-induced signaling cascade that promotes the chaperone activity of HspA1L with implications for understanding male reproductive biology.

The Link That Binds: The Linker of Hsp70 as a Helm of the Protein's Function.

The heat shock 70 (Hsp70) family of molecular chaperones plays a central role in maintaining cellular proteostasis. Structurally, Hsp70s are composed of an N-terminal nucleotide binding domain (NBD) which exhibits ATPase activity, and a C-terminal substrate binding domain (SBD). The binding of ATP at the NBD and its subsequent hydrolysis influences the substrate binding affinity of the SBD through allostery. Similarly, peptide binding at the C-terminal SBD stimulates ATP hydrolysis by the N-terminal NBD. Interdomain communication between the NBD and SBD is facilitated by a conserved linker segment. Hsp70s form two main subgroups. Canonical Hsp70 members generally suppress protein aggregation and are also capable of refolding misfolded proteins. Hsp110 members are characterized by an extended lid segment and their function tends to be largely restricted to suppression of protein aggregation. In addition, the latter serve as nucleotide exchange factors (NEFs) of canonical Hsp70s. The linker of the Hsp110 family is less conserved compared to that of the canonical Hsp70 group. In addition, the linker plays a crucial role in defining the functional features of these two groups of Hsp70. Generally, the linker of Hsp70 is quite small and varies in size from seven to thirteen residues. Due to its small size, any sequence variation that Hsp70 exhibits in this motif has a major and unique influence on the function of the protein. Based on sequence data, we observed that canonical Hsp70s possess a linker that is distinct from similar segments present in Hsp110 proteins. In addition, Hsp110 linker motifs from various genera are distinct suggesting that their unique features regulate the flexibility with which the NBD and SBD of these proteins communicate via allostery. The Hsp70 linker modulates various structure-function features of Hsp70 such as its global conformation, affinity for peptide substrate and interaction with co-chaperones. The current review discusses how the unique features of the Hsp70 linker accounts for the functional specialization of this group of molecular chaperones.

Suppression of hesA mutation on nitrogenase activity in Paenibacillus polymyxa WLY78 with the addition of high levels of molybdate or cystine.

The diazotrophic Paenibacillus polymyxa WLY78 possesses a minimal nitrogen fixation gene cluster consisting of nine genes (nifB nifH nifD nifK nifE nifN nifX hesA and nifV). Notably, the hesA gene contained within the nif gene cluster is also found within nif gene clusters among diazotrophic cyanobacteria and Frankia. The predicted product HesA is a member of the ThiF-MoeB-HesA family containing an N-terminal nucleotide binding domain and a C-terminal MoeZ/MoeB-like domain. However, the function of hesA gene in nitrogen fixation is unknown. In this study, we demonstrate that the hesA mutation of P. polymyxa WLY78 leads to nearly complete loss of nitrogenase activity. The effect of the mutation can be partially suppressed by the addition of high levels of molybdate or cystine. However, the nitrogenase activity of the hesA mutant could not be restored by Klebsiella oxytoca nifQ or Escherichia coli moeB completely. In addition, the hesA mutation does not affect nitrate reductase activity of P. polymyxa WLY78. Our results demonstrate hesA is a novel gene specially required for nitrogen fixation and its role is related to introduction of S and Mo into the FeMo-co of nitrogenase.

Structural analysis reveals pathomechanisms associated with pseudoxanthoma elasticum–causing mutations in the ABCC6 transporter

Mutations in ABC subfamily C member 6 (ABCC6) transporter are associated with pseudoxanthoma elasticum (PXE), a disease resulting in ectopic mineralization and affecting multiple tissues. A growing number of mutations have been identified in individuals with PXE. For most of these variants, no mechanistic information is available regarding their role in normal and pathophysiologies. To assess how PXE-associated mutations alter ABCC6 biosynthesis and structure, we biophysically and biochemically evaluated the N-terminal nucleotide-binding domain. A high-resolution X-ray structure of nucleotide-binding domain 1 (NBD1) of human ABCC6 was obtained at 2.3 Å that provided a template on which to evaluate PXE-causing mutations. Biochemical analysis of mutations in this domain indicated that multiple PXE-causing mutations altered its structural properties. Analyses of the full-length protein revealed a strong correlation between the alterations in NBD properties and the processing and expression of ABCC6. These results suggest that a significant fraction of PXE-associated mutations located in NBD1 causes changes in its structural properties and that these mutation-induced alterations directly affect the maturation of the full-length ABCC6 protein.

Mechanical properties of BiP protein determined by nano‐rheology

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (KD ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

Structures of the calcium-activated, non-selective cation channel TRPM4.

TRPM4 is a calcium-activated, phosphatidylinositol bisphosphate (PtdInsP2) modulated, non-selective cation channel, and belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the cryo-EM structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide binding domain (NBD) and the C-terminal coiled coil participate in the tetrameric assembly of the channel; ATP binds at NBD and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. S1-S4 domain and post-S6 TRP domain form the central gating apparatus that likely house the Ca2+ and PtdInsP2 binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and also reveal the molecular architecture of the TRPM family for the first time.

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle.

Genome-wide survey of heat shock factors and heat shock protein 70s and their regulatory network under abiotic stresses in Brachypodium distachyon.

The heat shock protein 70s (Hsp70s) and heat shock factors (Hsfs) play key roles in protecting plant cells or tissues from various abiotic stresses. Brachypodium distachyon, recently developed an excellent model organism for functional genomics research, is related to the major cereal grain species. Although B. distachyon genome has been fully sequenced, the information of Hsf and Hsp70 genes and especially the regulatory network between Hsfs and Hsp70s remains incomplete. Here, a total of 24 BdHsfs and 29 BdHsp70s were identified in the genome by bioinformatics analysis and the regulatory network between Hsfs and Hsp70s were performed in this study. Based on highly conserved domain and motif analysis, BdHsfs were grouped into three classes, and BdHsp70s divided into six groups, respectively. Most of Hsf proteins contain five conserved domains: DBD, HR-A/B region, NLS and NES motifs and AHA domain, while Hsp70 proteins have three conserved domains: N-terminal nucleotide binding domain, peptide binding domain and a variable C-terminal lid region. Expression data revealed a large number of BdHsfs and BdHsp70s were induced by HS challenge, and a previous heat acclimation could induce the acquired thermotolerance to help seedling suffer the severe HS challenge, suggesting that the BdHsfs and BdHsp70s played a role in alleviating the damage by HS. The comparison revealed that, most BdHsfs and BdHsp70s genes responded to multiple abiotic stresses in an overlapping relationship, while some of them were stress specific response genes. Moreover, co-expression relationships and predicted protein-protein interaction network implied that class A and B Hsfs played as activator and repressors, respectively, suggesting that BdHsp70s might be regulated by both the activation and the repression mechanisms under stress condition. Our genomics analysis of BdHsfs and BdHsp70s provides important evolutionary and functional characterization for further investigation of the accurate regulatory mechanisms among Hsfs and Hsp70s in herbaceous plants.

Structural Role of ABCG5/ABCG8 in Sterol Transport

ATP binding cassette (ABC) transporters play critical roles in maintaining sterol homeostasis in eukaryotic organisms, including yeast, plants and mammals. In humans, the heterodimeric ABCG5/ABCG8 (G5G8) mediates the excretion of cholesterol and dietary plant sterols into bile and into the gut lumen. Mutations inactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal recessive genetic disorder characterized by plant sterol accumulation, hypercholesterolemia, and premature coronary atherosclerosis. ABCG5 and ABCG8 are half ABC transporters;each subunit consists of an N-terminal nucleotide-binding domain (NBD) and a C-terminal transmembrane domain (TMD). The NBDs dimerize to form two catalytically asymmetric nucleotide-binding sites (NBS), one that is catalytically active (NBS2) and the other inactive (NBS1). To understand the structural basis for G5G8-mediated sterol transport we developed a large-scale purification of human G5G8 by exploiting Pichia patoris yeast. We crystallized the transporter in lipid bilayers, solved its structure in a nucleotide-free state at 3.9 Å resolution, and generated the first atomic model of an ABC sterol transporter. G5G8 presents a new structural configuration for the TMD of ABC transporters, which is present in a large and functionally diverse ABC2 superfamily. We discover that the TMD and the NBS are coupled through networks of interactions that differ between NBS1 and NBS2, reflecting the catalytic asymmetry of the transporter. A series of conserved polar residues in the TMD form polar networks that we proposed play a role in transmitting signals from the ATPase catalysis in the NBS to sterol transport on the TMD. Molecular dynamic simulation and long-range coevolution analysis revealed an inward-upward TMD movement that predicts a significant conformational change between the TMD subunits. Thus, the G5G8 structure provides a molecular framework that allows us to propose a mechanistic model for ABC transporter-mediated sterol transport and to analyze the disruptive effects of mutations causing sitosterolemia. The structure will serve as a structural template for homology modelling to a wide range of transport system that is regulated by ABCG transporters and by ABC2 superfamily.

Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.

Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures.