The chromosomal protein sso7d of the crenarchaeon Sulfolobus solfataricus rescues aggregated proteins in an ATP hydrolysis-dependent manner

In this work, we show that the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus displays a cation-dependent ATPase activity with a pH optimum around neutrality and a temperature optimum of 70 degrees C. Measurements of tryptophan fluorescence and experiments that used 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that ATP hydrolysis induces a conformational change in the molecule and that the binding of the nucleotide triggers the ATP hydrolysis-induced conformation of the protein to return to the native conformation. We found that Sso7d rescues previously aggregated proteins in an ATP hydrolysis-dependent manner; the native conformation of Sso7d forms a complex with the aggregates, while the ATP hydrolysis-induced conformation is incapable of this interaction. Sso7d is believed to be the first protein isolated from an archaeon capable of rescuing aggregates.     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.     

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3′-to-5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a “closed” state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an “open” state that differs by an ∼ 160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.     

Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains     

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.     

ATP induces conformational changes in the carboxyl-terminal region of ClC-5

ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation.     

Resolving individual steps in the operation of ATP-dependent proteolytic molecular machines: from conformational changes to substrate translocation and processivity

Clp, Lon, and FtsH proteases are proteolytic molecular machines that use the free energy of ATP hydrolysis to unfold protein substrates and processively present them to protease active sites. Here we review recent biochemical and structural studies relevant to the mechanism of ATP-dependent processive proteolysis. Despite the significant structural differences among the Clp, Lon, and FtsH proteases, these enzymes share important mechanistic features. In these systems, mechanistic studies have provided evidence for ATP binding and hydrolysis-driven conformational changes that drive translocation of substrates, which has significant implications for the processive mechanism of proteolysis. These studies indicate that the nucleotide (ATP, ADP, or nonhydrolyzable ATP analogues) occupancy of the ATPase binding sites can influence the binding mode and/or binding affinity for protein substrates. A general mechanism is proposed in which the communication between ATPase active sites and protein substrate binding regions coordinates a processive cycle of substrate binding, translocation, proteolysis, and product release.     

Crystal structure of tryptophanyl-tRNA synthetase complexed with adenosine-5' tetraphosphate: evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tRNA synthetases

Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5' tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the gamma and deltad phosphate groups to occupy positions equivalent to those occupied by the beta and gamma phosphates of ATP. The beta-phosphate effects a 1.11 A extension that relocates the alpha-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Palpha as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.     

Conformational state of the SecYEG-bound SecA probed by single tryptophan fluorescence spectroscopy

The SecYEG complex is a membrane-embedded channel that permits the passage of precursor proteins (preproteins) across the inner membrane of Escherichia coli. SecA is a molecular motor that associates with the SecYEG pore and drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the conformational state of soluble and SecYEG-bound SecA using single tryptophan mutants of SecA. The fluorescence spectral properties of the single tryptophans of SecA and their accessibility to the quencher acrylamide demonstrate that SecA undergoes a conformational change that results in a more compact structure upon binding of ATP and binding to the SecYEG pore. In addition, SecYEG-bound SecA undergoes ATP-dependent conformational changes that are not observed for soluble SecA. These data support a model in which binding to the SecYEG channel has a major impact on the SecA conformation.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Biochemical evidence for interaction between the two nucleotide binding domains of ArsA. Insights from mutants and ATP analogs

ArsA, the peripheral membrane component of the anion-translocating ATPase ArsAB, consists of two nucleotide binding domains (A1 and A2), which are connected by a linker sequence. Previous studies on ArsA have focused on the function of each nucleotide binding domain and the role of the linker, whereas the present study looks at the interactions between the binding domains and their interactions with the linker. It has previously been shown that the A1 domain of ArsA carries out unisite catalysis in the absence of antimonite, while A2 is recruited in multisite catalysis by antimonite in the presence of a functional A1 domain. Multisite catalysis thus seems to result from an interaction between A1 and A2 brought about by antimonite. In the present study, we provide direct biochemical evidence for interaction between the two nucleotide binding domains and show that the linker region acts as a transducer of the conformational changes between them. We find that nucleotide binding to the A2 domain results in a significant, detectable change in the conformation of the A1 domain. Two ATP analogs, FSBA and ATP gamma S, used in this study, were both found to bind preferentially to the A2 domain, and their binding resulted in changing the otherwise compact A1 domain into an open conformation. Point mutations in the A2 domain and the linker region also produced a similar effect on the conformation of A1, thus suggesting that events at A2 are relayed to A1 via the linker. We propose that nucleotide binding to A2 produces a two-tiered conformational change. The significance of these changes in the mechanism of ArsA is discussed.     

Nuclear magnetic resonance and fluorescence studies of substrate-induced conformational changes of histidine-binding protein J of Salmonella typhimurium.

The histidine-binding protein J of Salmonella typhimurium binds L-histidine as a first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution nuclear magnetic resonance spectroscopy has been used to monitor the conformation of histidine-binding protein J in the presence and absence of substrate. Evidence is presented to show that this binding protein undergoes a conformational change involving a substantial number of amino-acid residues (including tryptophans) in the presence of L-histidine and that this change is specific for L-histidine. In order to monitor the involvement of tryptophan residues in the substrate-induced conformational change, 5-fluorotryptophan has been incorporated biosynthetically into the histidine-binding protein J using a tryptophan autotroph of Salmonella typhimurium. There are no significant differences in the conformation and binding activity between the 5-fluorotryptophan-labeled and the normal histidine-binding protein J. Proton and fluorine-19 nuclear magnetic resonance studies of the 5-fluorotryptophan-labeled binding protein show that at least one (and possibly two) of the tryptophan residues undergo(es) a change toward a more hydrophobic environment in the presence of L-histidine. These observations are supported by fluorescence data and by differences in the reactivity of the tryptophan residues of this protein toward N-bromosuccinimide in the presence and absence of substrate. The present results are consistent with models for the action of periplasmic-binding proteins in shock-sensitive transport systems of gram-negative bacteria which require a substrate-induced conformational change prior to the energy-dependent translocation of substrates.     

Mapping interactions between the Ca2+-ATPase and its substrate ATP with infrared spectroscopy

Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2'-deoxy-ATP, 3'-deoxy-ATP, and inosine 5'-triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2'-OH, the 3'-OH, and the amino group of adenine reduce the extent of binding-induced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups. Phosphorylation of the ATPase was observed for ITP and 2'-deoxy-ATP, but not for 3'-deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa(2) also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.     

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.     

Characterization of the catalytic subunit of an anion pump

The ArsA protein, the 63-kDa catalytic subunit of an oxyanion-translocating ATPase, was purified by successive chromatography using Q-Sepharose, red agarose, and phenyl-Sepharose to a specific activity in excess of 1 mumol of ATP hydrolyzed per min per mg of protein. ATPase activity was dependent on the presence of the oxyanionic substrates. Inhibitors of other classes of ion-translocating ATPases had no effect on ArsA ATPase activity, including N,N'-dicyclohexyl-carbodiimide, azide, vanadate, and nitrate. The apparent Km for ATP was determined to be 0.13 mM. The optimal pH range for ATP hydrolysis was 7.5 to 7.8. ATPase activity required Mg2+ at a molar ratio of 2 ATP:1 Mg2+. Limited proteolysis by trypsin was used to study conformational changes produced upon binding of substrates to the ArsA protein. In the absence of substrates, the ArsA protein was rapidly cleaved by trypsin to a major product of 30 kDa. ATP was partially protected from trypsin digestion, while the anionic substrate antimonite alone had no effect on proteolysis. Combination of the two substrates nearly completely protected the ArsA protein from proteolysis. Proteolytic cleavage correlated with loss of anion-stimulated ATPase activity and substrate protection from cleavage correlated with retention of activity. These results demonstrate that ATP and antimonite together produce a conformational change which is different from that of the ArsA protein in the presence of either substrate alone and suggest interaction between the oxyanion and ATP binding sites.     

Conformational changes generated in GroEL during ATP hydrolysis as seen by time-resolved infrared spectroscopy

Changes in the vibrational spectrum of the chaperonin GroEL in the presence of ADP and ATP have been followed as a function of time using rapid scan Fourier transform infrared spectroscopy. The interaction of nucleotides with GroEL was triggered by the photochemical release of the ligands from their corresponding biologically inactive precursors (caged nucleotides; P3-1-(2-nitro)phenylethyl nucleotide). Binding of either ADP or ATP induced the appearance of small differential signals in the amide I band of the protein, sensitive to protein secondary structure, suggesting a subtle and localized change in protein conformation. Moreover, conformational changes associated with ATP hydrolysis were detected that differed markedly from those observed upon nucleotide binding. Both, high-amplitude absorbance changes and difference bands attributable to modifications in the interaction between oppositely charged residues were observed during ATP hydrolysis. Once this process had occurred, the protein relaxed to an ADP-like conformation. Our results suggest that the secondary structure as well as salt bridges of GroEL are modified during ATP hydrolysis, as compared with the ATP and ADP bound protein states.     

Structural changes in mitochondrial F1-ATPase induced by the removal of loosely bound nucleotides

Mitochondrial F1-ATPase from beef heart, forms aggregates when it is depleted of loosely bound nucleotides by repeated precipitation in ammonium sulfate. Polyacrylamide gradient gel electrophoresis, in non dissociating conditions shows that the aggregate formed is a dimer (708,000 daltons). The aggregation is attributed to a conformational change of the protein as a consequence of the elimination of the nucleotides from the low affinity binding sites. This structural alteration seems to be reversible because, after addition of ATP, the aggregation is not observed on polyacrylamide gels but the catalytic properties remain unchanged. This conformational change alters the accessibility of protein sulfhydryl groups to 5,5' - dithiobis(2-nitrobenzoic acid). All these observations emphasize the importance of protein nucleotide interactions to the conformation of the mitochondrial F1-ATPase.     

Conformational sampling and nucleotide-dependent transitions of the GroEL subunit probed by unbiased molecular dynamics simulations

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant "opening" of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition.