Spectroscopic and functional characterization of the [2Fe-2S] scaffold protein Nfu from Synechocystis PCC6803.

Iron-sulfur clusters are ubiquitous cofactors required for various essential metabolic processes. Conservation of proteins required for their biosynthesis and trafficking allows for simple bacteria to be used as models to aid in exploring these complex pathways in higher organisms. Cyanobacteria are among the most investigated organisms for these processes, as they are unicellular and can survive under photoautotrophic and heterotrophic conditions. Herein, we report the potential role of Synechocystis PCC6803 NifU (now named SyNfu) as the principal scaffold protein required for iron-sulfur cluster biosynthesis in that organism. SyNfu is a well-folded protein with distinct secondary structural elements, as evidenced by circular dichroism and a well-dispersed pattern of 1H-15N HSQC NMR peaks, and readily reconstitutes as a [2Fe-2S] dimeric protein complex. Cluster exchange experiments show that glutathione can extract the cluster from holo-SyNfu, but the transfer is unidirectional. We also confirm the ability of SyNfu to transfer cluster to both human ferredoxin 1 and ferredoxin 2, while also demonstrating the capacity to deliver cluster to both monothiol glutaredoxin 3 and dithiol glutaredoxin 2. This evidence supports the hypothesis that SyNfu indeed serves as the main scaffold protein in Synechocystis, as it has been shown to be the only protein required for viability in the absence of photoautotrophic conditions. Similar to other NFU-type cluster donors and other scaffold and carrier proteins, such as ISCU, SyNfu is shown by DSC to be structurally less stable than regular protein donors, while retaining a relatively well-defined tertiary structure as represented by 1H-15N HSQC NMR experiments.

Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein.

Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly.

Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography-mass spectrometry (LC-MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.

Defining the mechanism of the mitochondrial Atm1p [2Fe-2S] cluster exporter.

Iron-sulfur cluster proteins play key roles in a multitude of physiological processes; including gene expression, nitrogen and oxygen sensing, electron transfer, and DNA repair. Biosynthesis of iron-sulfur clusters occurs in mitochondria on iron-sulfur cluster scaffold proteins in the form of [2Fe-2S] cores that are then transferred to apo targets within metabolic or respiratory pathways. The mechanism by which cytosolic Fe-S cluster proteins mature to their holo forms remains controversial. The mitochondrial inner membrane protein Atm1p can transport glutathione-coordinated iron-sulfur clusters, which may connect the mitochondrial and cytosolic iron-sulfur cluster assembly systems. Herein we describe experiments on the yeast Atm1p/ABCB7 exporter that provide additional support for a glutathione-complexed cluster as the natural physiological substrate and a reflection of the endosymbiotic model of mitochondrial evolution. These studies provide insight on the mechanism of cluster transport and the molecular basis of human disease conditions related to ABCB7. Recruitment of MgATP following cluster binding promotes a structural transition from closed to open conformations that is mediated by coupling helices, with MgATP hydrolysis facilitating the return to the closed state.

Characterization of [2Fe-2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods.

Many iron-sulfur proteins involved in cluster trafficking form [2Fe-2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe-2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA-like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe-2S] species, such as (FDX2:GLRX5:[2Fe-2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe-2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron-sulfur-cluster-bridged protein complexes and transient iron-sulfur-cluster transfer intermediates.

Reconstitution, characterization, and [2Fe-2S] cluster exchange reactivity of a holo human BOLA3 homodimer.

A new class of mitochondrial disease has been identified and characterized as Multiple Mitochondrial Dysfunctions Syndrome (MMDS). Four different forms of the disease have each been attributed to point mutations in proteins involved in iron-sulfur (Fe-S) biosynthesis; in particular, MMDS2 has been associated with the protein BOLA3. To date, this protein has been characterized in vitro concerning its ability to form heterodimeric complexes with two putative Fe-S cluster-binding partners: GLRX5 and NFU. However, BOLA3 has yet to be characterized in its own discrete holo form. Herein we describe procedures to isolate and characterize the human holo BOLA3 protein in terms of Fe-S cluster binding and trafficking and demonstrate that human BOLA3 can form a functional homodimer capable of engaging in Fe-S cluster transfer.

Understanding the Mechanism of [4Fe-4S] Cluster Assembly on Eukaryotic Mitochondrial and Cytosolic Aconitase.

Iron-sulfur (Fe-S) clusters are common prosthetic groups that are found within a variety of proteins responsible for functions that include electron transfer, regulation of gene expression, and substrate binding and activation. Acquisition of a [4Fe-4S] cluster is essential for the functionality of many such roles, and dysfunctions in Fe-S cluster synthesis and trafficking often result in human disease, such as multiple mitochondrial dysfunctions syndrome. While the topic of [2Fe-2S] cluster biosynthesis and trafficking has been relatively well studied, the understanding of such processes involving [4Fe-4S] centers is less developed. Herein, we focus on the mechanism of the assembly of [4Fe-4S] clusters on two members of the aconitase family, differing also in organelle placement (mitochondrion and cytosol) and biochemical function. Two mechanistic models are evaluated by a combination of kinetic and spectroscopic models, namely, a consecutive model (I), in which two [2Fe-2S] clusters are sequentially delivered to the target, and a prereaction equilibrium model (II), in which a [4Fe-4S] cluster transiently forms on a donor protein before transfer to the target. The paper also addresses the issue of cluster nuclearity for functionally active forms of ISCU, NFU, and ISCA trafficking proteins, each of which has been postulated to exist in both [2Fe-2S] and [4Fe-4S] bound states. By the application of kinetic assays and electron paramagnetic resonance spectroscopy to examine delivery pathways from a variety of potential [2Fe-2S] donor proteins to eukaryotic forms of both aconitase and iron regulatory protein, we conclude that a consecutive model following the delivery of [2Fe-2S] clusters from NFU1 is the most likely mechanism for these target proteins.

Cluster exchange reactivity of [2Fe-2S] cluster-bridged complexes of BOLA3 with monothiol glutaredoxins.

The [2Fe-2S] cluster-bridged complex of BOLA3 with GLRX5 has been implicated in cluster trafficking, but cluster exchange involving this heterocomplex has not been reported. Herein we describe an investigation of the cluster exchange reactivity of holo BOLA3-GLRX complexes using two different monothiol glutaredoxins, H.s. GLRX5 and S.c. Grx3, which share significant identity. We observe that a 1 : 1 mixture of apo BOLA3 and glutaredoxin protein is able to accept a cluster from donors such as ISCU and a [2Fe-2S](GS)4 complex, with preferential formation of the cluster-bridged heterodimer over the plausible holo homodimeric glutaredoxin. Holo BOLA3-GLRX5 transfers clusters to apo acceptors at rates comparable to other Fe-S cluster trafficking proteins. Isothermal titration calorimetry experiments with apo proteins demonstrated a strong binding of BOLA3 with both GLRX5 and Grx3, while binding with an alternative mitochondrial partner, NFU1, was weak. Cluster exchange and calorimetry experiments resulted in a very similar behavior for yeast Grx3 (cytosolic) and human GLRX5 (mitochondrial), indicating conservation across the monothiol glutaredoxin family for interactions with BOLA3 and supporting a functional role for the BOLA3-GLRX5 heterocomplex relative to the previously proposed BOLA3-NFU1 interaction. The results also demonstrate rapid formation of the heterocomplexed holo cluster via delivery from a glutathione-complexed cluster, again indicative of the physiological relevance of the [2Fe-2S](GS)4 complex in the cellular labile iron pool.

Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex.

Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examined the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those observed for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Additionally, the chaperone complex was able to promote Nfu function by enhancing the second-order rate constants for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking.

In Vitro Studies of Cellular Iron-Sulfur Cluster Biosynthesis, Trafficking, and Transport.

Iron-sulfur clusters are metal cofactors that comprise the largest class of metalloproteins and are utilized for a wide variety of functions ranging from electron transport to DNA repair. These clusters and their respective cluster-binding proteins are highly conserved and are produced in the mitochondria via an evolutionarily conserved process for export to the cytosol and delivery to other organelles, including the nucleus. Disruption of the biosynthetic pathway results in a number of disease conditions that reflect the essential requirements of cluster function and trafficking within the cell. In vivo studies are limited in their ability to examine the detailed molecular mechanisms of protein-protein interactions, since they often focus on the downstream effects of protein depletion or mutation. As such, in vitro analyses are essential for defining the roles of specific Fe-S proteins in trafficking events and supporting in vivo analyses of disease conditions arising from aberrant Fe-S assembly and trafficking. In this chapter, we describe a variety of methods for the analysis of structure-function relationships in holo Fe-S cluster proteins, as well as monitoring the kinetics and molecular mechanisms of Fe-S cluster transfer.

Analysis of NFU-1 metallocofactor binding-site substitutions-impacts on iron-sulfur cluster coordination and protein structure and function.

Iron-sulfur (Fe/S) clusters are ancient prosthetic groups found in numerous metalloproteins and are conserved across all kingdoms of life due to their diverse, yet essential functional roles. Genetic mutations to a specific subset of mitochondrial Fe/S cluster delivery proteins are broadly categorized as disease-related under multiple mitochondrial dysfunction syndrome (MMDS), with symptoms indicative of a general failure of the metabolic system. Multiple mitochondrial dysfunction syndrome 1 (MMDS1) arises as a result of the missense mutation in NFU1, an Fe/S cluster scaffold protein, which substitutes a glycine near the Fe/S cluster-binding pocket to a cysteine (p.Gly208Cys). This substitution has been shown to promote protein dimerization such that cluster delivery to NFU1 is blocked, preventing downstream cluster trafficking. However, the possibility of this additional cysteine, located adjacent to the cluster-binding site, serving as an Fe/S cluster ligand has not yet been explored. To fully understand the consequences of this Gly208Cys replacement, complementary substitutions at the Fe/S cluster-binding pocket for native and Gly208Cys NFU1 were made, along with six other variants. Herein, we report the results of an investigation on the effect of these substitutions on both cluster coordination and NFU1 structure and function. The data suggest that the G208C substitution does not contribute to cluster binding. Rather, replacement of the glycine at position 208 changes the oligomerization state as a result of global structural alterations that result in the downstream effects manifest as MMDS1, but does not perturb the coordination chemistry of the Fe-S cluster.

Understanding the molecular basis for multiple mitochondrial dysfunctions syndrome 1 (MMDS1): impact of a disease-causing Gly189Arg substitution on NFU1.

Iron-sulfur (Fe/S) cluster-containing proteins constitute one of the largest protein classes, with highly varied function. Consequently, the biosynthesis of Fe/S clusters is evolutionarily conserved and mutations in intermediate Fe/S cluster scaffold proteins can cause disease, including multiple mitochondrial dysfunctions syndrome (MMDS). Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (p.Gly189Arg) near the active site of NFU1, an Fe/S scaffold protein. In vitro investigation into the structure-function relationship of the Gly189Arg derivative, along with two other variants, reveals that substitution at position 189 triggers structural changes that increase flexibility, decrease stability, and alter the monomer-dimer equilibrium toward monomer, thereby impairing the ability of the Gly189X derivatives to receive an Fe/S cluster from physiologically relevant sources.

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)-Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway.

Iron-sulfur (Fe/S)-cluster-containing proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an Fe/S scaffold protein, via an in vitro investigation into the structural and functional consequences. Analysis of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure composition. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an Fe/S cluster from physiologically relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change.

Cytosolic iron-sulfur cluster transfer-a proposed kinetic pathway for reconstitution of glutaredoxin 3.

Iron-sulfur (Fe-S) clusters are ubiquitously conserved and play essential cellular roles. The mechanism of Fe-S cluster biogenesis involves multiple proteins in a complex pathway. Cluster biosynthesis primarily occurs in the mitochondria, but key Fe-S proteins also exist in the cytosol. One such protein, glutaredoxin 3 (Grx3), is involved in iron regulation, sensing, and mediating [2Fe-2S] cluster delivery to cytosolic protein targets, but the cluster donor for cytosolic Grx3 has not been elucidated. Herein, we delineate the kinetic transfer of [2Fe-2S] clusters into Grx3 from potential cytosolic carrier/scaffold proteins, IscU and Nfu, to evaluate a possible model for Grx3 reconstitution in vivo.

Mapping cellular Fe-S cluster uptake and exchange reactions - divergent pathways for iron-sulfur cluster delivery to human ferredoxins.

Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe-2S] or [4Fe-4S] clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe-2S] cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular cluster trafficking. This is the first step toward building a network map for all of the possible iron-sulfur cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

Glutathione-complexed [2Fe-2S] clusters function in Fe-S cluster storage and trafficking.

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS4) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe-S cluster transfer reactions. UV-vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS4) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS4) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe-S cluster proteins, thereby supporting a possible physiological role for such centers.

Iron-sulfur cluster exchange reactions mediated by the human Nfu protein.

Human Nfu is an iron-sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron-sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe-2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe-2S](GS)4 cluster complex, and extraction of the Nfu-bound iron-sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe-2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe-2S] cluster trafficking and delivery.

Inactivation of sortase A mediated by metal ATCUN complexes.

Catalytic metallopeptides that target the membrane-associated sortase A transpeptidase have been developed and evaluated as irreversible inactivators of SrtA∆N59 (sortase A, lacking the initial membrane-binding domain). The copper-binding GGH tripeptide ATCUN motif was linked to amidated forms of the cell wall sorting signal, LPET and LPETG, as sortase-targeting moieties. The resulting metallopeptides were used to determine half maximal inhibitory concentrations (IC50) and rate constants for time-dependent sortase A inactivation. Michaelis-Menten behavior was observed for the catalytic metallopeptides, and k(cat), K(M) and k(cat)/K(M) parameters were obtained as 0.080 ± 0.002 min⁻¹, 23 ± 2 µM and 0.0035 ± 0.0003 µM⁻¹ min⁻¹, respectively. Concentration-dependent inhibition of SrtA∆N59 by the metallopeptides revealed IC50 values ranging from 570 to 700 µM, while Cu-GGH, which lacked a targeting motif, had no measurable IC50 value (>2,000 µM). Time-dependent inactivation of SrtA revealed a range of catalytic activities, with Cu-GGHGLPETG-NH2 demonstrating the fastest rate of inactivation in the presence of ascorbate and hydrogen peroxide coreactants. The active site of the enzyme comprises residues Cys-184, Arg-197 and His-120. LC-MS/MS analysis of the reaction products demonstrated modification of Cys-184 to cysteine sulfonic acid (+48 amu). Results obtained from a DTNB assay support oxidation of the Cys-184 residue. LC-MS/MS also suggested oxidation of the Arg-197 containing peptide. 2D NMR analysis was performed to assess the possible oxidation of His-120, however, none was observed. These compounds possess the potential for irreversible inactivation of SrtA through oxidative modification of essential residues required for substrate binding.

Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone.

Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain. We determined the crystal structure of the NBD of human Mortalin at 2.8 Å resolution. Although the Mortalin nucleotide-binding pocket is highly conserved relative to other Hsp70 family members, we find that its nucleotide affinity is weaker than that of Hsc70. A Parkinson's disease-associated mutation is located on the Mortalin-NBD surface and may contribute to Mortalin aggregation. We present structure-based models for how the Mortalin-NBD may interact with the nucleotide exchange factor GrpEL1, with p53, and with MKT-077. Our structure may contribute to the understanding of disease-associated Mortalin mutations and to improved Mortalin-targeting antitumor compounds.