Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.     

D1 ring is stable and nucleotide-independent,whereas D2 ring undergoes major conformational changes during the ATPase cycle of p97-VCP

The 97-kDa valosin-containing protein (p97-VCP) belongs to the AAA (ATPases associated with various cellular activities) family and acts as a molecular chaperone in diverse cellular events, including ubiquitinproteasome-mediated degradation. We previously showed that VCP contains a substrate-binding domain, N, and two conserved ATPase domains, D1 and D2, of which D2 is responsible for the major enzyme activity. VCP has a barrel-like structure containing two stacked homo-hexameric rings made of the D1 and D2 domains, and this structure is essential for its biological functions. During ATPase cycles, VCP undergoes conformational changes that presumably apply tensions to the bound substrate, leading to the disassembly of protein complexes or unfolding of the substrate. How ATPase activity is coupled with the conformational changes in VCP complex and the D1 and D2 rings is not clear. In this report, we took biochemical approaches to study the structure of VCP in different nucleotide conditions to depict the conformational changes in the ATPase cycles. In contrast to many AAA chaperones that require ATP/ADP to form oligomers, both wild type VCP and ATP-binding site mutants can form hexamers without the addition of nucleotide. This nucleotide-independent hexamerization requires an intact D1 and the down-stream linker sequence of VCP. Tryptophan fluorescence and trypsin digestion analyses showed that ATP/ADP binding induces dramatic conformational changes in VCP. These changes do not require the presence of an intact ATP-binding site in D1 and is thus mainly attributed to the D2 domain. We propose a model whereby D1, although undergoing minor conformational changes, remains as a relatively trypsin-resistant hexameric ring throughout the ATPase cycle, whereas D2 only does so when it binds to ATP or ADP. After ADP is released at the end of the ATP hydrolysis, D2 ring is destabilized and adopts a relatively flexible and open structure.     

Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. Different patterns in the two nucleotide binding domains

The function of the human cystic fibrosis transmembrane conductance regulator (CFTR) protein as a chloride channel or transport regulator involves cellular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide occlusion, detected by covalent labeling with 8-azido-[alpha-32P]ATP. Nucleotide occlusion in CFTR requires incubation at 37 degrees C, and the occluded nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the labeled CFTR protein we found that the adenine nucleotide occlusion preferentially occurred in the N-terminal nucleotide binding domain (NBD). Addition of the ATPase inhibitor vanadate, which stabilizes an open state of the CFTR chloride channel, produced an increased nucleotide occlusion and resulted in the labeling of both the N-terminal and C-terminal NBDs. Protein modification with N-ethylmaleimide prevented both vanadate-dependent and -independent nucleotide occlusion in CFTR. The pattern of nucleotide occlusion indicates significant differences in the ATP hydrolyzing activities of the two NBDs, which may explain their different roles in the CFTR channel regulation.     

ATP-induced structural transitions in PAN, the proteasome-regulatory ATPase complex in Archaea

ATP binding to the PAN-ATPase complex in Archaea or the homologous 19 S protease-regulatory complex in eukaryotes induces association with the 20 S proteasome and opening of its substrate entry channel, whereas ATP hydrolysis allows unfolding of globular substrates. To clarify the conformational changes associated with ATP binding and hydrolysis, we used protease sensitivity to monitor the conformations of the PAN ATPase from Methanococcus jannischii. Exhaustive trypsin treatment of PAN generated five distinct fragments, two of which differed when a nucleotide (either ATP, ATP gamma S, or ADP) was bound. Surprisingly, the nucleotide concentrations altering protease sensitivity were much lower (K(a) 20-40 microm) than are required for ATP-dependent protein breakdown by the PAN-20S proteasome complex (K(m) approximately 300-500 microm). Unlike trypsin, proteinase K yielded several fragments that differed in the ATP gamma S and ADP-bound forms, and thus revealed conformational transitions associated with ATP hydrolysis. Mapping the fragments generated by each revealed that nucleotide binding and hydrolysis induce local conformational changes, affecting the Walker A and B nucleotide-binding motif, as well as global changes extending to its carboxyl terminus. The location and overlap of the fragments also suggest that the conformation of the six subunits is not identical, probably because they do not all bind ATP simultaneously. Partial nucleotide occupancy was supported by direct assays, which demonstrated that, at saturating conditions, only four nucleotides are bound to hexameric PAN. Using the protease protection maps, we modeled the conformational changes associated with ATP binding and hydrolysis in PAN based on the x-ray structures of the homologous AAA ATPase, HslU.     

Purification and ATPase activity of human ABCA1

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.     

Interaction of the nucleotide binding domains and regulation of the ATPase activity of the human retina specific ABC transporter, ABCR

We report here a novel regulation of the ATPase activity of the human retina specific ATP binding cassette transporter (ABC), ABCR, by nucleotide binding domain interactions. We also present evidence that recombinant nucleotide binding domains of ABCR interact in vitro in the complete absence of transmembrane domains (TMDs). Although similar domain-domain interactions have been described in other ABC transporters, the roles of such interactions on the enzymatic mechanisms of these transporters have not been demonstrated experimentally. A quantitative analysis of the in vitro interactions as a function of the nucleotide-bound state demonstrated that the interaction takes place in the absence of nucleotide as well as in the presence of ATP and that it only attenuates in the ADP-bound state. Analysis of the ATPase activities of these proteins in free and complex states indicated that the NBD1-NBD2 interaction significantly influences the ATPase activity. Further investigation, using site-specific mutants, showed that mutations in NBD2 but not NBD1 led to the alteration of the ATPase activity of the NBD1.NBD2 complex and residue Arg 2038 is critical to this regulation. These data indicate that changes in the oligomeric state of the nucleotide binding domains of ABCR are coupled to ATP hydrolysis and might represent a possible signal for the TMDs of ABCR to export the bound substrate. Furthermore, the data support a mechanistic model in which, upon binding of NBD2, NBD1 binds ATP but does not hydrolyze it or does so with a significantly reduced rate.     

A structural pathway for activation of the kinesin motor ATPase

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.     

Stability of the topoisomerase II closed clamp conformation may influence DNA-stimulated ATP hydrolysis

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.     

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.     

Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.     

Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter

The rod outer segment (ROS) ABC transporter (ABCR) plays an important role in the outer segment of retinal rod cells, where it functions as a transporter of all-trans retinal, most probably as the complex lipid, retinylidene-phosphatidyl-ethanolamine. We report here a quantitative analysis of the structural and functional effects of genetic mutations, associated with several macular degenerations, in the second nucleotide-binding domain of ABCR (NBD2). We have analyzed the ATP binding, kinetics of ATP hydrolysis, and structural changes. The results of these multifaceted analyses were correlated with the disease severity and prognosis. Results presented here demonstrated that, in wild type NBD2, distinct conformational changes accompany nucleotide (ATP and ADP) binding. Upon ATP binding, NBD2 protein changed to a relaxed conformation where tryptophans became more solvent-exposed, while ADP binding reverses this process and leads back to a taut conformation that is also observed with the unbound protein. This sequence of conformational change appears to be important in the energetics of the ATP hydrolysis and may have important structural consequences in the ability of the NBD2 domain to act as a regulator of the nucleotide-binding domain 1. Some of the mutant proteins displayed strikingly different patterns of conformational changes upon nucleotide binding that pointed to unique structural consequences of these genetic mutations. The ABCR dysfunctions, associated with various retinopathies, are multifaceted in nature and include alterations in protein structure as well as the attenuation of ATPase activity and nucleotide binding.     

The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins

The action of the molecular chaperone Hsp90 is essential for the activation and assembly of an increasing number of client proteins. This function of Hsp90 has been proposed to be governed by conformational changes driven by ATP binding and hydrolysis. Association of co-chaperones and client proteins regulate the ATPase activity of Hsp90. Here, we have examined the inhibition of the ATPase activity of human Hsp90beta by one such co-chaperone, human p23. We demonstrate that human p23 interacts with Hsp90 in both the absence and presence of nucleotide with a higher affinity in the presence of the ATP analogue AMP-PNP. This is consistent with an analysis of the effect of p23 on the steady-state kinetics that revealed a mixed mechanism of inhibition. Mass spectrometry of the intact Hsp90.p23 complex determined the stoichiometry of binding to be one p23 to each subunit of the Hsp90 dimer. p23 was also shown to interact with a monomeric, truncated fragment of Hsp90, lacking the C-terminal homodimerisation domain, indicating dimerisation of Hsp90 is not a prerequisite for association with p23. Complex formation between Hsp90 and p23 increased the apparent affinity of Hsp90 for AMP-PNP and completely inhibited the ATPase activity. We propose a model where the role of p23 is to lock individual subunits of Hsp90 in an ATP-dependent conformational state that has a high affinity for client proteins.     

ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.     

Structural dissection of ATP turnover in the prototypical GHL ATPase TopoVI

GHL proteins are functionally diverse enzymes defined by the presence of a conserved ATPase domain that self-associates to trap substrate upon nucleotide binding. The structural states adopted by these enzymes during nucleotide hydrolysis and product release, and their consequences for enzyme catalysis, have remained unclear. Here, we have determined a complete structural map of the ATP turnover cycle for topoVI-B, the ATPase subunit of the archaeal GHL enzyme topoisomerase VI. With this ensemble of structures, we show that significant conformational changes in the subunit occur first upon ATP binding, and subsequently upon release of hydrolyzed P(i). Together, these data provide a structural framework for understanding the role of ATP hydrolysis in the type II topoisomerase reaction. Our results also suggest that the GHL ATPase module is a molecular switch in which ATP hydrolysis serves as a prerequisite but not a driving force for substrate-dependent structural transitions in the enzyme.     

Characterization of Cdr1p, a major multidrug efflux protein of Candida albicans: purified protein is amenable to intrinsic fluorescence analysis

Candida drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, confers multidrug resistance in immunocompromised and debilitated patients. A member of the ATP-binding cassette (ABC) superfamily of membrane transporters, Cdr1p contains two nucleotide binding/utilization sites (NBDs) and two transmembrane domains (TMDs). We had earlier characterized Cdr1p by its overexpression as a GFP-tagged fusion protein that elicits oligomycin-sensitive ATPase activity and is linked to drug extrusion. However, it is essential to have highly purified Cdr1p to understand the detailed molecular basis of structure and functions of this protein. In this study, we have developed a two-step purification protocol using stably overexpressed His-tagged Cdr1p in Saccharomyces cerevisiae. Purified Cdr1p exhibited divalent cation-dependent ATPase activity [approximately 1.2 micromol (mg of protein)(-)(1) min(-)(1)] with an apparent K(M) in the range of 1.8 to 2.1 mM and V(max) between 1.0 and 1.4 micromol (mg of protein)(-)(1) min(-)(1). Unlike its close homologue human P-gp/MDR1, purified Cdr1p only moderately displayed drug stimulated ATPase activity. By exploiting intrinsic fluorescence intensity of purified Cdr1p, which contains 24 tryptophan residues, we could monitor defined conformational changes upon substrate drug and ATP binding. It is observed that ATP binding to Cdr1p (K(d) = approximately 1.7 mM) is not a prerequisite for drug binding, and both the mechanisms of drug as well as ATP binding, which induce specific conformational changes, occur independent of each other. Our study for the first time provides a catalytically active purified ABC transporter from a fungal pathogen, which is amenable to fluorescence measurements and thus would be useful in understanding the molecular basis of antifungal transport.     

Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones

GRP94, an essential endoplasmic reticulum chaperone, is required for the conformational maturation of proteins destined for cell-surface display or export. The extent to which GRP94 and its cytosolic paralog, Hsp90, share a common mechanism remains controversial. GRP94 has not been shown conclusively to hydrolyze ATP or bind cochaperones, and both activities, by contrast, result in conformational changes and N-terminal dimerization in Hsp90 that are critical for its function. Here, we report the 2.4 A crystal structure of mammalian GRP94 in complex with AMPPNP and ADP. The chaperone is conformationally insensitive to the identity of the bound nucleotide, adopting a "twisted V" conformation that precludes N-terminal domain dimerization. We also present conclusive evidence that GRP94 possesses ATPase activity. Our observations provide a structural explanation for GRP94's observed rate of ATP hydrolysis and suggest a model for the role of ATP binding and hydrolysis in the GRP94 chaperone cycle.     

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.     

Independent ATPase activity of Hsp90 subunits creates a flexible assembly platform

The ATPase activity of the molecular chaperone Hsp90 is essential for its function in the assembly of client proteins. To understand the mechanism of human Hsp90, we have carried out a detailed kinetic analysis of ATP binding, hydrolysis and product release. ATP binds rapidly in a two-step process involving the formation of a diffusion-collision complex followed by a conformational change. The rate-determining step was shown to be ATP hydrolysis and not subsequent ADP dissociation. There was no evidence from any of the biophysical measurements for cooperativity in either nucleotide binding or hydrolysis for the dimeric protein. A monomeric fragment, lacking the C-terminal dimerisation domain, showed no dependence on protein concentration and, therefore, subunit association for activity. The thermodynamic linkage between client protein binding and nucleotide affinity revealed ATP bound Hsp90 has a higher affinity for client proteins than the ADP bound form. The kinetics are consistent with independent Michaelis-Menten catalysis in each subunit of the Hsp90 dimer. We propose that Hsp90 functions in an open-ring configuration for client protein activation.     

Human ABCA3, a product of a responsible gene for abca3 for fatal surfactant deficiency in newborns, exhibits unique ATP hydrolysis activity and generates intracellular multilamellar vesicles

ABCA3 is highly expressed at the membrane of lamellar bodies in alveolar type II cells, in which pulmonary surfactant is stored. ABCA3 gene mutations cause fatal surfactant deficiency in newborns. We established HEK293 cells stably expressing human ABCA3 and analyzed the function. Exogenously expressed ABCA3 is glycosylated and localized at the intracellular vesicle membrane. ABCA3 is efficiently photoaffinity labeled by 8-azido-[alpha(32)P]ATP, but not by 8-azido-[gamma(32)P]ATP, when the membrane fraction is incubated in the presence of orthovanadate. Photoaffinity labeling of ABCA3 shows unique metal ion-dependence and is largely reduced by membrane pretreatment with 5% methyl-beta-cyclodextrin, which depletes cholesterol. Electron micrographs show that HEK293/hABCA3 cells contain multivesicular, lamellar body-like structures, which do not exist in HEK293 host cells. Some fuzzy components such as lipids accumulate in the vesicles. These results suggest that ABCA3 shows ATPase activity, which is induced by lipids, and may be involved in the biogenesis of lamellar body-like structures.     

Chemical modification of the Ca2+-dependent ATPase of sarcoplasmic reticulum from skeletal muscle. I. Binding of N-ethylmaleimide to sarcoplasmic reticulum: evidence for sulfhydryl groups in the active site of ATPase and for conformational changes induced by adenosine tri- and diphosphate.

The time course of binding of N-ethylmaleimide (NEM) to the SR was measured at pH 7.5 in the presence or absence of ATP or ADP. The following results were obtained. 1. Both in the presence and absence of nucleotide, the ATPase [EC 3.6.1.3] activity decreased linearly with increase in the amount of NEM bound to the fragmented sarcoplasmic reticulum (SR), and was inhibited almost completely by the binding of 2 moles of NEM per 10(5) g of the SR protein. 2. The amount of NEM incorporated into the ATPase (M.W.=105,000) was measured by SDS disc-gel electrophoresis. It was shown that the ATPase activity was inhibited almost completely by the binding of 2 moles of NEM per mole of ATPase. 3. The rate of binding of NEM to SR decreased by 30-40% in the presence of either ATP or ADP. The concentrations of both ATP and ADP for half-saturation were 0.1-0.2mM. 4. The effect of nucleotide on the rate of binding of NEM was not changed by the presence of Ca2+ and Mg2+ ions. Similar effects were also observed even when the SR membranes were solubilized with Triton X-100. It is suggested from these results that one or two SH groups are located in the active site of the SR ATPase, and that conformational changes are induced by the addition of ATP and ADP.