The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines.

ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.

BY-kinases: Protein tyrosine kinases like no other.

BY-kinases (for bacterial tyrosine kinases) constitute a family of protein tyrosine kinases that are highly conserved in the bacterial kingdom and occur most commonly as essential components of multicomponent assemblies responsible for the biosynthesis, polymerization, and export of complex polysaccharides involved in biofilm or capsule formation. BY-kinase function has been attributed to a cyclic process involving formation of an oligomeric species, its disassembly into constituent monomers, and subsequent reassembly, depending on the overall phosphorylation level of a C-terminal cluster of tyrosine residues. However, the relationship of this process to the active/inactive states of the enzyme and the mechanism of its integration into the polysaccharide production machinery remain unclear. Here, we synthesize the substantial body of biochemical, cell-biological, structural, and computational data, acquired over the nearly 3 decades since the discovery of BY-kinases, to suggest means by which they fulfill their physiological function. We propose a mechanism involving temporal coordination of the assembly/disassembly process with the autokinase activity of the enzyme and its ability to be dephosphorylated by its counteracting phosphatase. We speculate that this temporal control enables BY-kinases to function as molecular timers that coordinate the diverse processes involved in the synthesis, polymerization, and export of complex sugar derivatives. We suggest that BY-kinases, which deploy distinctive catalytic domains resembling P-loop nucleoside triphosphatases, have uniquely adapted this ancient fold to drive functional processes through exquisite spatiotemporal control over protein-protein interactions and conformational changes. It is our hope that the hypotheses proposed here will facilitate future experiments targeting these unique protein kinases.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes.

The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in αßα sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an α-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first αßα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.

Decoding the Conformational Selective Mechanism of FGFR Isoforms: A Comparative Molecular Dynamics Simulation.

Fibroblast growth factor receptors (FGFRs) play critical roles in the regulation of cell growth, differentiation, and proliferation. Specifically, FGFR2 gene amplification has been implicated in gastric and breast cancer. Pan-FGFR inhibitors often cause large toxic side effects, and the highly conserved ATP-binding pocket in the FGFR1/2/3 isoforms poses an immense challenge in designing selective FGFR2 inhibitors. Recently, an indazole-based inhibitor has been discovered that can selectively target FGFR2. However, the detailed mechanism involved in selective inhibition remains to be clarified. To this end, we performed extensive molecular dynamics simulations of the apo and inhibitor-bound systems along with multiple analyses, including Markov state models, principal component analysis, a cross-correlation matrix, binding free energy calculation, and community network analysis. Our results indicated that inhibitor binding induced the phosphate-binding loop (P-loop) of FGFR2 to switch from the open to the closed conformation. This effect enhanced extensive hydrophobic FGFR2-inhibitor contacts, contributing to inhibitor selectivity. Moreover, the key conformational intermediate states, dynamics, and driving forces of this transformation were uncovered. Overall, these findings not only provided a structural basis for understanding the closed P-loop conformation for therapeutic potential but also shed light on the design of selective inhibitors for treating specific types of cancer.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1-843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.

The Local Environment of Loop Switch 1 Modulates the Rate of ATP-Induced Dissociation of Human Cardiac Actomyosin.

Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster upon ATP binding, and the affinity of ADP to actomyosin is weaker. One can suggest that the isoform-specific actomyosin kinetics is regulated at the nucleotide binding site of human cardiac myosin. Myosin is a P-loop ATPase; the nucleotide-binding site consists of P-loop and loops switch 1 and 2. All three loops position MgATP for successful hydrolysis. Loops sequence is conserved in both myosin isoforms, and we hypothesize that the isoform-specific structural element near the active site regulates the rate of nucleotide binding and release. Previously we ran molecular dynamics simulations and found that loop S291-E317 near loop switch 1 is more compact and exhibits larger fluctuations of the position of amino acid residues in beta isoform than in alpha. In alpha isoform, the loop forms a salt bridge with loop switch 1, the bridge is not present in beta isoform. Two isoleucines I303 and I313 of loop S291-E317 are replaced with valines in alpha isoform. We introduced a double mutation I303V:I313V in beta isoform background and studied how the mutation affects the rate of ATP binding and ADP dissociation from actomyosin. We found that ATP-induced actomyosin dissociation occurs faster in the mutant, but the rate of ADP release remains the same as in the wild-type beta isoform. Due to the proximity of loop S291-E317 and loop switch 1, a faster rate of ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site. A similar rate of ADP dissociation from actomyosin in the mutant and wild-type myosin constructs indicates that loop switch 1 does not control ADP release from actomyosin.

Analyses of the oncogenic BRAFD594G variant reveal a kinase-independent function of BRAF in activating MAPK signaling.

Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF), that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies have reported that kinase-dead BRAF variants amplify mitogen-activated protein kinase (MAPK) signaling by dimerizing with and activating WT C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared with WT BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the αC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium toward the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major players in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Last, we propose that the dimer interface of the BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.

Fungal Kti12 proteins display unusual linker regions and unique ATPase p-loops.

Kti12 (Kluyveromyces lactis toxin insensitive 12) is an evolutionary highly conserved ATPase, crucial for the tRNA-modification activity of the eukaryotic Elongator complex. The protein consists of an N-terminal ATPase and a C-terminal tRNA-binding domain, which are connected by a flexible linker. The precise role of the linker region and its involvement in the communication between the two domains and their activities remain elusive. Here, we analyzed all available Kti12 protein sequences and report the discovery of a subset of Kti12 proteins with abnormally long linker regions. These Kti12 proteins are characterized by a co-occurring lysine to leucine substitution in their Walker A motif, previously thought to be invariable. We show that the K14L substitution lowers the affinity to ATP, but does not affect the catalytic activity of Kti12 at high ATP concentrations. We compare the activity of mutated variants of Kti12 in vitro with complementation assays in vivo in yeast. Ultimately, we compared Kti12 to other known p-loop ATPase family members known to carry a similar deviant Walker A motif. Our data establish Kti12 of Eurotiomycetes as an example of eukaryotic ATPase harboring a significantly deviating but still functional Walker A motif.

Structural insights into the G-loop dynamics of E. coli FtsY NG domain.

The bacterial signal recognition particle (SRP) receptor, FtsY, participates with the SRP in co-translation targeting of proteins. Multiple crystal structures of the NG domain of E. coli FtsYNG have been determined at high-resolution (1.22-1.88 Å), in the nucleotide-free (apo) form as well as bound to GDP and non-hydrolysable GTP analogues. The combination of high-resolution and multiple solved structures of FtsYNG in different states revealed a new sensor-relay system of this unique GTPase receptor. A nucleotide sensing function of the P-loop assists FtsYNG in nucleotide-binding and contributes to modulate nucleotide binding properties in SRP GTPases. A reorganization of the other G-loops and the insertion binding domain (IBD) is observed only upon transition from a diphosphate to a triphosphate nucleotide. The role of a magnesium ion during the GDP and GTP-bound states has also been observed. The binding of magnesium in the nucleotide site causes the reorientation of the ß- and γ- phosphate groups toward the jaws of the P-loop and stabilizes the binding of the nucleotide, creating a network of hydrogen and water-bridge interactions.

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.

G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life.

Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1 complex of the A1AO ATP synthase.

F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A1AO ATP synthases (A3B3CDE2FG2ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A3B3D- and A3B3DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments.

Plant STAND P-loop NTPases: a current perspective of genome distribution, evolution, and function : Plant STAND P-loop NTPases: genomic organization, evolution, and molecular mechanism models contribute broadly to plant pathogen defense.

STAND P-loop NTPase is the common weapon used by plant and other organisms from all three kingdoms of life to defend themselves against pathogen invasion. The purpose of this study is to review comprehensively the latest finding of plant STAND P-loop NTPase related to their genomic distribution, evolution, and their mechanism of action. Earlier, the plant STAND P-loop NTPase known to be comprised of only NBS-LRRs/AP-ATPase/NB-ARC ATPase. However, recent finding suggests that genome of early green plants comprised of two types of STAND P-loop NTPases: (1) mammalian NACHT NTPases and (2) NBS-LRRs. Moreover, YchF (unconventional G protein and members of P-loop NTPase) subfamily has been reported to be exceptionally involved in biotic stress (in case of Oryza sativa), thereby a novel member of STAND P-loop NTPase in green plants. The lineage-specific expansion and genome duplication events are responsible for abundance of plant STAND P-loop NTPases; where "moderate tandem and low segmental duplication" trajectory followed in majority of plant species with few exception (equal contribution of tandem and segmental duplication). Since the past decades, systematic research is being investigated into NBS-LRR function supported the direct recognition of pathogen or pathogen effectors by the latest models proposed via 'integrated decoy' or 'sensor domains' model. Here, we integrate the recently published findings together with the previous literature on the genomic distribution, evolution, and distinct models proposed for functional molecular mechanism of plant STAND P-loop NTPases.

How RNase R Degrades Structured RNA: ROLE OF THE HELICASE ACTIVITY AND THE S1 DOMAIN.

RNase R, a ubiquitous 3' exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on single-stranded RNA.

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system.

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1-D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.

Biogenesis of cytosolic and nuclear iron-sulfur proteins and their role in genome stability.

Iron-sulfur (Fe-S) clusters are versatile protein cofactors that require numerous components for their synthesis and insertion into apoproteins. In eukaryotes, maturation of cytosolic and nuclear Fe-S proteins is accomplished by cooperation of the mitochondrial iron-sulfur cluster (ISC) assembly and export machineries, and the cytosolic iron-sulfur protein assembly (CIA) system. Currently, nine CIA proteins are known to specifically assist the two major steps of the biogenesis reaction. They are essential for cell viability and conserved from yeast to man. The essential character of this biosynthetic process is explained by the involvement of Fe-S proteins in central processes of life, e.g., protein translation and numerous steps of nuclear DNA metabolism such as DNA replication and repair. Malfunctioning of these latter Fe-S enzymes leads to genome instability, a hallmark of cancer. This review is focused on the maturation and biological function of cytosolic and nuclear Fe-S proteins, a topic of central interest for both basic and medical research. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016.

The P-loop region of Schlafen 3 acts within the cytosol to induce differentiation of human Caco-2 intestinal epithelial cells.

Schlafen 3 (Slfn3) mediates rodent enterocyte differentiation in vitro and in vivo, required for intestinal function. Little is known about Schlafen protein structure-function relationships. To define the Slfn3 domain that promotes differentiation, we studied villin and sucrase isomaltase (SI) promoter activity in Slfn3-null human Caco-2BBE cells transfected with full-length rat Slfn3 DNA or truncated constructs. Confocal microscopy and Western blots showed that Slfn3 is predominantly cytosolic. Villin promoter activity, increased by wild type Slfn3, was further enhanced by adding a nuclear exclusion sequence, suggesting that Slfn3 does not affect transcription by direct nuclear action. We therefore sought to dissect the region in Slfn3 stimulating promoter activity. Since examination of the Slfn3 N-terminal region revealed sequences similar to both an aminopeptidase (App) and a divergent P-loop resembling those in NTPases, we initially divided Slfn3 into an N-terminal domain containing the App and P-loop regions, and a C-terminal region. Only the N-terminal construct stimulated promoter activity. Further truncation indicated that both the App and the smaller P-loop constructs enhanced promoter activity similarly to the N-terminal sequence. Point mutations within the N-terminal region (R128L, altering a critical active site residue in the App domain, and L212D, conserved in Schlafens but variable in P-loop proteins) did not affect activity. These results show that Slfn3 acts in the cytosol to trigger a secondary signal cascade that elicits differentiation marker expression and narrows the active domain to the third of the Slfn3 sequence homologous to P-loop NTPases, a first step in understanding its mechanism of action.

Crystal structure of Legionella pneumophila dephospho-CoA kinase reveals a non-canonical conformation of P-loop.

Dephospho-CoA kinase (DPCK; EC 2.7.1.24) catalyzes the final step in the coenzyme A biosynthetic pathway. DPCK transfers a phosphate group from ATP to the 3-hydroxyl group of the ribose of dephosphocoenzyme A (dCoA) to yield CoA and ADP. Upon the binding of ligands, large conformational changes is induced in DPCKs, as well as in many other kinases, to shield the bound ATP in their catalytic site from the futile hydrolysis by bulk water molecules. To investigate the molecular mechanisms underlying the phosphoryl transfer during DPCK catalytic cycle, we determined the crystal structures of the Legionellapneumophila DPCK (LpDPCK) both in its apo-form and in complex with ATP. The structures reveal that LpDPCK comprises of three domains, the classical core domain, the CoA domain, and the LID domain, which are packed together to create a central cavity for substrate-binding and enzymatic catalysis. The binding of ATP induces large conformational changes, including a hinge-bending motion of the CoA binding domain and the "helix to loop" conformational change of the P-loop. Finally, modeling of a dCoA molecule to the enzyme provides insights into the catalytic mechanism of DPCK.

Interaction with Cfd1 increases the kinetic lability of FeS on the Nbp35 scaffold.

P-loop NTPases of the ApbC/Nbp35 family are involved in FeS protein maturation in nearly all organisms and are proposed to function as scaffolds for initial FeS cluster assembly. In yeast and animals, Cfd1 and Nbp35 are homologous P-loop NTPases that form a heterotetrameric complex essential for FeS protein maturation through the cytosolic FeS cluster assembly (CIA) pathway. Cfd1 is conserved in animals, fungi, and several archaeal species, but in many organisms, only Nbp35 is present, raising the question of the unique roles played by Cfd1 and Nbp35. To begin to investigate this issue, we examined Cfd1 and Nbp35 function in budding yeast. About half of each protein was detected in a heterocomplex in logarithmically growing yeast. Nbp35 readily bound (55)Fe when fed to cells, whereas (55)Fe binding by free Cfd1 could not be detected. Rapid (55)Fe binding to and release from Nbp35 was impaired by Cfd1 deficiency. A Cfd1 mutation that caused a defect in heterocomplex stability supported iron binding to Nbp35 but impaired iron release. Our results suggest a model in which Cfd1-Nbp35 interaction increases the lability of assembled FeS on the Nbp35 scaffold for transfer to target apo-FeS proteins.