Intramyocardial Transplantation of Human iPS Cell-Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals.

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.

Structural Basis of Activin Receptor-Like Kinase 2 (R206H) Inhibition by Bis-heteroaryl Pyrazole-Based Inhibitors for the Treatment of Fibrodysplasia Ossificans Progressiva Identified by the Integration of Ligand-Based and Structure-Based Drug Design Approaches.

Fibrodysplasia ossificans progressiva (FOP) is a rare but severe genetic disorder in which acute inflammation elicits progressive heterotopic ossification in the muscles, tendons, and ligaments. Classic FOP is caused by the R206H mutation in ALK2/ACVR1. While several activin receptor-like kinase 2 (ALK2) inhibitors were found to be efficacious in animal models of FOP, most of the ALK2 (R206H) inhibitors lacked sufficient oral bioavailability for efficacy. Previously, the synthesis of a series of novel bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitors that achieved both substantial potency and an improved ADMET profile was reported. In the present study, the detailed procedure of the in silico approach employed to identify the initial bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitor RK-59638 and the analysis of the ALK2 (R206H) RK-59638 complex structure to guide the synthetic optimization of the chemical series, obtaining RK-71807 showing improved potency and metabolic stability, were described. According to the initial in silico screening, the screening efficiencies and chemical diversity of the hit compounds of both ligand-based and structure-based methods were evaluated. Then, X-ray structures of ALK2 (R206H) and the inhibitors were analyzed to assess the structure-activity relationships of the synthesized compounds. The 3D-RISM analysis indicated the existence of the additional hydrogen bond via water molecules restricting the attachment point in the pyrazole scaffold. The quantum mechanics calculation of the newly determined ALK2 (R206H) RK-71807 complex structure using a fragment molecular orbital method and pair interaction energy decomposition analysis was employed to evaluate the interaction energies between the inhibitor and each of the amino acid residues and decompose them to electrostatic, exchange-repulsion, and charge transfer energies. The pattern of decomposed interaction energies was then compared to that formed by RK-59638 and LDN-193189 to investigate the structural basis of ALK2 (R206H) inhibition.

Protein ligand interaction analysis against new CaMKK2 inhibitors by use of X-ray crystallography and the fragment molecular orbital (FMO) method.

CaMKK2 (calcium/calmodulin dependent protein kinase kinase 2) is a serine/threonine protein kinase that regulates phosphorylation of CaM kinases (CaMKs) such as CaMKI, CaMKIV, and AMP-activated protein kinase (AMPK). From a pathological perspective, CaMKK2 plays a role in obesity, diabetes, and prostate cancer. Therefore, CaMKK2 is an attractive target protein for drug design. Here, we tried to find new CaMKK2 inhibitors by using ligand-based and structure-based drug design approaches. From the in silico hit compounds, we identified new inhibitors by using a CaMKK2 kinase assay. We solved X-ray crystallography structures of the CaMKK2-inhibitor complexes and performed Fragment Molecular Orbital (FMO) calculations to analyze the protein-ligand interactions, identify the key residues in inhibitor binding, and quantitatively measure their contribution. We experimentally determined five CaMKK2-inhibitor structures and calculated the binding energies of the inhibitors by the FMO method plus MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) approach. The results showed a high correlation (R = -0.89) between experimentally measured inhibitory activity (pIC50) and the predicted ligand binding energy. We then quantitatively evaluated the contribution of each binding site residue in CaMKK2 by the IFIE (Inter-fragment Interaction Energy)/PIEDA (Pair Interaction Energy Decomposition Analysis) method. The IFIE values indicated that Lys194 and Glu236, which formed hydrogen bonds with the carboxylate groups of the inhibitors, were key residues for ligand binding. PIEDA revealed that the dispersion interaction of inhibitors with hydrophobic residues, such as Ile171, Phe267, and Leu319, contributed highly to ligand binding; we considered that this was due to CH-π interactions with methoxy groups and/or aromatic rings contained in our CaMKK2 inhibitor. These results from the quantitative interaction analysis by the FMO method are useful not only for future CaMMK2 inhibitor development but for application of the FMO method to in silico drug design.

Development and Structural Evaluation of N-Alkylated trans-2-Phenylcyclopropylamine-Based LSD1 Inhibitors.

Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the demethylation of histone H3 and regulates gene expression. Because it is implicated in the regulation of diseases such as acute myeloid leukemia, potent LSD1-specific inhibitors have been pursued. Trans-2-phenylcyclopropylamine (2-PCPA)-based inhibitors featuring substitutions on the amino group have emerged, with sub-micromolar affinities toward LSD1 and high selectivities over monoamine oxidases (MAOs). We synthesized two N-alkylated 2-PCPA-based LSD1 inhibitors, S2116 and S2157, based on the previously developed S2101. S2116 and S2157 exhibited enhanced potency for LSD1 by 2.0- to 2.6-fold, as compared with S2101. In addition, they exhibited improved selectivity over MAOs. Structural analyses of LSD1 co-crystallized with S2101, S2116, S2157, or another N-alkylated inhibitor (FCPA-MPE) confirmed that the N-substituents enhance the potency of a 2-PCPA-based inhibitor of LSD1, without constituting the adduct formed with FAD.

Bis-Heteroaryl Pyrazoles: Identification of Orally Bioavailable Inhibitors of Activin Receptor-Like Kinase-2 (R206H).

Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.

Development of a transplant injection device for optimal distribution and retention of human induced pluripotent stem cell‒derived cardiomyocytes.

BACKGROUND: Induced pluripotent stem cell (iPSC)‒based regenerative therapy is a promising strategy for cardiovascular disease treatment; however, the method is limited by the myocardial retention of grafted iPSCs. Thus, an injection protocol that efficiently introduces and retains human iPSC-derived cardiomyocytes (hiPSC-CMs) within the myocardium is urgently needed. The objective of the present study was to develop a method to improve the retention of hiPSCs in the myocardium for cardiac therapy. METHODS: We efficiently produced hiPSC-CM spheroids in 3-dimensional (3D) culture using microwell plates, and developed an injection device for optimal 3D distribution of the spheroids in the myocardial layer. Device biocompatibility was assessed with purified hiPSC-CM spheroids. Device effectiveness was evaluated in 10- to 15-month-old farm pigs (n = 15) and 5- to 24-month-old micro-minipigs (n = 20). The pigs were euthanized after injection, and tissues were harvested for retention and histologic analysis. RESULTS: We demonstrated an injection device for direct intramyocardial transplantation of hiPSC-CM spheroids from large-scale culture. The device had no detrimental effects on cell viability, spheroid shape, or size. Direct epicardial injection of spheroids mixed with gelatin hydrogel into beating porcine hearts using this device resulted in better distribution and retention of transplanted spheroids in a layer within the myocardium than did conventional needle injection procedures. CONCLUSIONS: The combination of the newly developed transplant device and spheroid formation promotes the retention of transplanted CMs. These findings support the clinical application of hiPSC-CM spheroid‒based cardiac regenerative therapy in patients with heart failure.

Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription.

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

Crystal structures of the S6K1 kinase domain in complexes with inhibitors.

Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.

Kinase crystal identification and ATP-competitive inhibitor screening using the fluorescent ligand SKF86002.

The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.

A pyrrolo-pyrimidine derivative targets human primary AML stem cells in vivo.

Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

Crystal structure of the guanylate kinase domain from discs large homolog 1 (DLG1/SAP97).

Discs large homolog 1 (DLG1/SAP97) is involved in the development and regulation of neuronal and immunological synapses. DLG1 is a member of the membrane associated guanylate kinase (MAGUK) family of proteins, which function as molecular scaffolds. The C-terminal guanylate kinase (GK) domain of DLG1 binds peptides with a phosphorylated serine residue. In this study, we solved the crystal structure of the GK domain of human DLG1. The C-terminal tail of DLG1 is bound to the peptide-binding site of an adjacent symmetry-related DLG1 GK molecule. The binding direction of the C-terminal tail to the peptide-binding site is opposite to that of the phosphorylated LGN peptide in complex with the rat DLG1 GK domain. The C-terminal tail forms a 310 helix, which is also different from the conformation of the phosphorylated LGN peptide. Nevertheless, the side chain interactions of the C-terminal tail with the DLG1 GK domain are similar to those of the phosphorylated LGN peptide.

Structures of histone methyltransferase SET7/9 in complexes with adenosylmethionine derivatives.

SET7/9 is a protein lysine methyltransferase that methylates histone H3 and nonhistone proteins such as p53, TAF10 and oestrogen receptor α. In previous work, novel inhibitors of SET7/9 that are amine analogues of the coenzyme S-(5'-adenosyl)-L-methionine (AdoMet) have been developed. Here, crystal structures of SET7/9 are reported in complexes with two AdoMet analogues, designated DAAM-3 and AAM-1, in which an n-hexylaminoethyl group or an n-hexyl group is attached to the N atom that replaces the S atom of AdoMet, respectively. In both structures, the inhibitors bind to the coenzyme-binding site and their additional alkyl chain binds in the lysine-access channel. The N atom in the azaalkyl chain of DAAM-3 is located at almost the same position as the N-methyl C atom of the methylated lysine side chain in the substrate-peptide complex structures and stabilizes complex formation by hydrogen bonding to the substrate-binding site residues of SET7/9. On the other hand, the alkyl chain of AAM-1, which is a weaker inhibitor than DAAM-3, binds in the lysine-access channel only through hydrophobic and van der Waals interactions. Unexpectedly, the substrate-binding site of SET7/9 complexed with AAM-1 specifically interacts with the artificial N-terminal sequence of an adjacent symmetry-related molecule, presumably stabilizing the alkyl chain of AAM-1.

Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor.

The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012), J. Med. Chem. 55, 5151-5156]. The report described the process of optimization of the structure-activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the ß3 strand located above the binding site, is not usually observed in Pim-1 structures.

A novel Pim-1 kinase inhibitor targeting residues that bind the substrate peptide.

A new screening method using fluorescent correlation spectroscopy was developed to select kinase inhibitors that competitively inhibit the binding of a fluorescently labeled substrate peptide. Using the method, among approximately 700 candidate compounds selected by virtual screening, we identified a novel Pim-1 kinase inhibitor targeting its peptide binding residues. X-ray crystal analysis of the complex structure of Pim-1 with the inhibitor indicated that the inhibitor actually binds to the ATP-binding site and also forms direct interactions with residues (Asp128 and Glu171) that bind the substrate peptide. These interactions, which cause small side-chain movements, seem to affect the binding ability of the fluorescently labeled substrate. The compound inhibited Pim-1 kinase in vitro, with an IC(50) value of 150 nM. Treatment of cultured leukemia cells with the compound reduced the amount of p21 and increased the amount of p27, due to Pim-1 inhibition, and then triggered apoptosis after cell-cycle arrest at the G(1)/S phase. This screening method may be widely applicable for the identification of various new Pim-1 kinase inhibitors targeting the residues that bind the substrate peptide.

Crystal structures of the armadillo repeat domain of adenomatous polyposis coli and its complex with the tyrosine-rich domain of Sam68.

Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68 kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm and Sam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.

ZF21 protein, a regulator of the disassembly of focal adhesions and cancer metastasis, contains a novel noncanonical pleckstrin homology domain.

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cells We have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, ß-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to ß-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.

Biochemical and structural studies on the high affinity of Hsp70 for ADP.

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the ß-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.

Structural basis for compound C inhibition of the human AMP-activated protein kinase α2 subunit kinase domain.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.

An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol, and other triterpenoids from Inonotus obliquus.

An unusual lanostane-type triterpenoid, spiroinonotsuoxodiol (1), and two lanostane-type triterpenoids, inonotsudiol A (2) and inonotsuoxodiol A (3), were isolated from the sclerotia of Inonotus obliquus. Their structures were determined to be (3S,7S,9R)-3,7-dihydroxy-7(8-->9)abeo-lanost-24-en-8-one (1), lanosta-8,24-dien-3beta,11beta-diol (2), and (22R)-3beta,22-dihydroxylanosta-8,24-dien-11-one (3) on the basis of NMR spectroscopy, including 1D and 2D ((1)H-(1)H COSY, NOESY, HMQC, HMBC) NMR, and FABMS. Compounds 1-3 showed moderate activity against cultured P388, L1210, HL-60 and KB cells.

The C-terminal BAG domain of BAG5 induces conformational changes of the Hsp70 nucleotide-binding domain for ADP-ATP exchange.

ADP-ATP exchange by the molecular chaperone Hsp70 is enhanced by several cochaperones. BAG5 consists of five BAG domains and associates with the nucleotide-binding domain (NBD) of Hsp70. The overexpression of BAG5 in the cytosol reportedly disturbs Hsp70-mediated protein refolding and induces Parkinson's disease. In the present study, we found that the fifth BAG domain (BD5) of BAG5 is responsible for the interaction between Hsp70 and BAG5. We also determined the crystal structures of the BD5*NBD complex. BD5 binding caused two different types of NBD conformational changes, which both disrupted the nucleotide-binding groove. In fact, BD5 reduced the affinity of the NBD for ADP. Moreover, BD5, as well as the full-length BAG5, accelerated Hsp70-mediated refolding in an in vitro assay. Therefore, BAG5 can function as the nucleotide exchange factor of Hsp70 for the enhancement of protein refolding.