ABSTRACT
KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.
Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms, Experimental/drug therapy , Piperazines/pharmacology , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Quinazolines/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Proliferation/drug effects , Cells, Cultured , Female , HCT116 Cells , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Docking Simulation , Mutation , Piperazines/chemistry , Piperazines/therapeutic use , Protein Binding , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Quinazolines/chemistry , Quinazolines/therapeutic useABSTRACT
Proteins are dynamic molecules; they undergo crucial conformational changes induced by post-translational modifications and by binding of cofactors or other molecules. The characterization of these conformational changes and their relation to protein function is a central goal of structural biology. Unfortunately, most conventional methods to obtain structural information do not provide information on protein dynamics. Therefore, mass spectrometry-based approaches, such as limited proteolysis, hydrogen-deuterium exchange, and stable-isotope labeling, are frequently used to characterize protein conformation and dynamics, yet the interpretation of these data can be cumbersome and time consuming. Here, we present PepShell, a tool that allows interactive data analysis of mass spectrometry-based conformational proteomics studies by visualization of the identified peptides both at the sequence and structure levels. Moreover, PepShell allows the comparison of experiments under different conditions, including different proteolysis times or binding of the protein to different substrates or inhibitors.
Subject(s)
Data Display , Mass Spectrometry/methods , Protein Conformation , Proteins/chemistry , Proteomics/methods , SoftwareABSTRACT
Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.
Subject(s)
Isotope Labeling/methods , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Proteomics/methods , Proto-Oncogene Proteins B-raf/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Deuterium Exchange Measurement , Humans , Indoles/chemistry , Indoles/pharmacology , MAP Kinase Signaling System/drug effects , Mass Spectrometry/methods , Molecular Dynamics Simulation , Mutation , Niacinamide/analogs & derivatives , Niacinamide/chemistry , Niacinamide/pharmacology , Peptides/analysis , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Phosphorylation/drug effects , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Proteolysis , Proteomics/instrumentation , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Sorafenib , Sulfonamides/chemistry , Sulfonamides/pharmacology , Trypsin/chemistry , VemurafenibABSTRACT
Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21(WAF1/CIP1), an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies.
Subject(s)
Brain Neoplasms/enzymology , Brain Neoplasms/pathology , DNA Replication , Glioblastoma/enzymology , Glioblastoma/pathology , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological , Animals , Cell Line, Tumor , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Breaks, Double-Stranded , Gene Knockdown Techniques , Histones/metabolism , Mice , Models, Biological , Phenotype , Retinoblastoma Protein/metabolism , S Phase , Signal Transduction , Tumor Suppressor Protein p53/metabolism , Up-RegulationABSTRACT
The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.
Subject(s)
Protein Kinase Inhibitors , Proto-Oncogene Proteins B-raf , Vemurafenib , Protein Kinase Inhibitors/chemistry , Molecular Dynamics Simulation , Mutation , Cell Line, TumorABSTRACT
The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.
Subject(s)
Enzyme Inhibitors/therapeutic use , Lung Neoplasms/drug therapy , Protein-Arginine N-Methyltransferases/drug effects , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Animals , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Humans , Lung Neoplasms/pathology , Mice , Pyrimidines/pharmacology , Pyrroles/pharmacologyABSTRACT
Protein arginine methyltransferase 5 (PRMT5) is an enzyme that can symmetrically dimethylate arginine residues in histones and nonhistone proteins by using S-adenosyl methionine (SAM) as the methyl donating cofactor. We have designed a library of SAM analogues and discovered potent, cell-active, and selective spiro diamines as inhibitors of the enzymatic function of PRMT5. Crystallographic studies confirmed a very interesting binding mode, involving protein flexibility, where both the cofactor pocket and part of substrate binding site are occupied by these inhibitors.
ABSTRACT
Targeted inhibition of protein kinases with small molecule drugs has evolved into a viable approach for anticancer therapy. However, the true selectivity of these therapeutic agents has remained unclear. Here, we used a proteomic method to profile the cellular targets of the clinical epidermal growth factor receptor kinase inhibitor gefitinib. Our data suggest alternative cellular modes of action for gefitinib and provide rationales for the development of related drugs.
Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Animals , COS Cells , Chlorocebus aethiops , ErbB Receptors/antagonists & inhibitors , Gefitinib , HeLa Cells , Humans , Proteomics/methods , Structure-Activity RelationshipABSTRACT
Knowledge about molecular drug action is critical for the development of protein kinase inhibitors for cancer therapy. Here, we establish a chemical proteomic approach to profile the anticancer drug SU6668, which was originally designed as a selective inhibitor of receptor tyrosine kinases involved in tumor vascularization. By employing immobilized SU6668 for the affinity capture of cellular drug targets in combination with mass spectrometry, we identified previously unknown targets of SU6668 including Aurora kinases and TANK-binding kinase 1. Importantly, a cell cycle block induced by SU6668 could be attributed to inhibition of Aurora kinase activity. Moreover, SU6668 potently suppressed antiviral and inflammatory responses by interfering with TANK-binding kinase 1-mediated signal transmission. These results show the potential of chemical proteomics to provide rationales for the development of potent kinase inhibitors, which combine rather unexpected biological modes of action by simultaneously targeting defined sets of both serine/threonine and tyrosine kinases involved in cancer progression.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Indoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , Animals , Aurora Kinases , COS Cells , Cell Division/drug effects , Chlorocebus aethiops , HeLa Cells , Humans , Oxindoles , Propionates , Protein Serine-Threonine Kinases/antagonists & inhibitors , TransfectionABSTRACT
The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.
Subject(s)
B-Lymphocytes/metabolism , Phosphoproteins/genetics , Proteome/genetics , Receptor, ErbB-3/genetics , Receptor, ErbB-4/genetics , Signal Transduction , Amino Acid Sequence , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Cell Line , Gene Expression Regulation , Gene Regulatory Networks , Genetic Engineering , Humans , Mice , Molecular Sequence Data , Neuregulin-1/metabolism , Neuregulin-1/pharmacology , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Interaction Mapping , Proteome/metabolism , Receptor, ErbB-3/metabolism , Receptor, ErbB-4/metabolismABSTRACT
The V600E missense mutation in B-Raf kinase leads to an anomalous regulation of the MAPK pathway, uncontrolled cell proliferation, and initiation of tumorigenesis. While the ATP-competitive B-Raf inhibitors block the MAPK pathway in B-Raf mutant cells, they induce conformational changes to wild-type B-Raf kinase domain leading to heterodimerization with C-Raf causing a paradoxical hyperactivation of MAPK pathway. A new class of inhibitors (paradox breakers) has been developed that inhibit B-Raf(V600E) activity without agonistically affecting the MAPK pathway in wild-type B-Raf cells. In this study, we explore the structural, conformational, and cellular effects on the B-Raf kinase domain upon binding of paradox breakers and inducers. Our results indicate that a subtle structural difference between paradox inducers and breakers leads to significant conformational differences when complexed with B-Raf. This study provides a novel insight into the activation of B-Raf by ATP-competitive inhibitors and can aid in the design of more potent and selective inhibitors without agonistic function.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity RelationshipABSTRACT
Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.
Subject(s)
Azepines/administration & dosage , Benzamides/administration & dosage , Breast Neoplasms/genetics , DNA Damage/drug effects , Enzyme Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/biosynthesis , Apoptosis/drug effects , Ataxia Telangiectasia Mutated Proteins/biosynthesis , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/radiotherapy , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Forkhead Box Protein M1 , Forkhead Transcription Factors/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/geneticsABSTRACT
Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 µM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology.
ABSTRACT
A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK.
ABSTRACT
Small-molecule inhibitors of protein kinases constitute a novel class of drugs for therapeutic intervention in a variety of human diseases. Most of these agents target the relatively conserved ATP-binding site of protein kinases and have only been tested against a rather small subset of all human protein kinases. Therefore, the selectivity of protein kinase inhibitors has remained a widely underestimated, but highly important issue in drug development programs. In this review, we focus on the recent advancement of chemical proteomic methods to evaluate drug selectivity in an unbiased, comprehensive way. Efficient affinity purification procedures using immobilized kinase inhibitors combined with the sensitivity of mass spectrometry detection permit the mapping of drug targets on a proteome-wide scale. Data from this type of assessment can be used to set up tailor-made selectivity panels, which guide compound development in the context of the most relevant off-targets during lead optimization. In cases in which identified alternative targets are of validated clinical relevance, chemical proteomics provides the opportunity to repeatedly exploit a once established kinase inhibitor principle for additional target kinases and can thereby dramatically shorten the time toward highly selective, preclinical candidates. Moreover, the identification of alternative targets for preclinical or clinical drugs can provide new insights into their cellular modes of action, which might help to define those disease settings in which the most beneficial therapeutic effect is likely to occur.
Subject(s)
Enzyme Inhibitors/pharmacology , Protein Kinase Inhibitors , Proteomics/methods , Animals , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/chemistry , Enzymes, Immobilized/chemistry , Humans , Protein Kinases/metabolismABSTRACT
UNLABELLED: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-ß3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE: CD74NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.
Subject(s)
Adenocarcinoma, Mucinous/genetics , Adenocarcinoma/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , Histocompatibility Antigens Class II/genetics , Lung Neoplasms/genetics , Neuregulin-1/genetics , Oncogene Proteins, Fusion/genetics , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Adenocarcinoma, Mucinous/pathology , Adult , Aged , Aged, 80 and over , Animals , Base Sequence , Cell Line, Tumor , Female , Gene Expression Profiling , Humans , Lung Neoplasms/pathology , Male , Mice , Middle Aged , Molecular Sequence Data , NIH 3T3 Cells , Oncogene Proteins, Fusion/metabolism , Sequence Analysis, DNA , Signal Transduction/geneticsABSTRACT
The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.