A Computational Analysis of ATP Binding of SV40 Large Tumor Antigen Helicase Motor

Simian Virus 40 Large Tumor Antigen (LTag) is an efficient helicase motor that unwinds and translocates DNA. The DNA unwinding and translocation of LTag is powered by ATP binding and hydrolysis at the nucleotide pocket between two adjacent subunits of an LTag hexamer. Based on the set of high-resolution hexameric structures of LTag helicase in different nucleotide binding states, we simulated a conformational transition pathway of the ATP binding process using the targeted molecular dynamics method and calculated the corresponding energy profile using the linear response approximation (LRA) version of the semi-macroscopic Protein Dipoles Langevin Dipoles method (PDLD/S). The simulation results suggest a three-step process for the ATP binding from the initial interaction to the final tight binding at the nucleotide pocket, in which ATP is eventually “locked” by three pairs of charge-charge interactions across the pocket. Such a “cross-locking” ATP binding process is similar to the binding zipper model reported for the F1-ATPase hexameric motor. The simulation also shows a transition mechanism of Mg²⁺ coordination to form the Mg-ATP complex during ATP binding, which is accompanied by the large conformational changes of LTag. This simulation study of the ATP binding process to an LTag and the accompanying conformational changes in the context of a hexamer leads to a refined cooperative iris model that has been proposed previously.     

Rotary catalysis of the stator ring of F1-ATPase

F₁-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α₃β₃ ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F₁-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Single-Molecule Imaging Reveals the Mechanism Underlying Histone Loading of Schizosaccharomyces pombe AAA+ ATPase Abo1

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.     

Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation

Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.     

Rotary catalysis of the stator ring of F(1)-ATPase

F(1)-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α(3)β(3) ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a "dictator" completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F(1)-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen

The large tumor antigen (LTag) of simian virus 40, an AAA(+) protein, is a hexameric helicase essential for viral DNA replication in eukaryotic cells. LTag functions as an efficient molecular machine powered by ATP binding and hydrolysis for origin DNA melting and replication fork unwinding. To understand how ATP binding and hydrolysis are coupled to conformational changes, we have determined high-resolution structures ( approximately 1.9 A) of LTag hexamers in distinct nucleotide binding states. The structural differences of LTag in various nucleotide states detail the molecular mechanisms of conformational changes triggered by ATP binding/hydrolysis and reveal a potential mechanism of concerted nucleotide binding and hydrolysis. During these conformational changes, the angles and orientations between domains of a monomer alter, creating an "iris"-like motion in the hexamer. Additionally, six unique beta hairpins on the channel surface move longitudinally along the central channel, possibly serving as a motor for pulling DNA into the LTag double hexamer for unwinding.     

Dynamic and coordinating domain motions in the active subunits of the F1-ATPase molecular motor

F1-ATPase is a rotary molecular motor crucial for various cellular functions. In F1-ATPase, the rotation of the gammadeltaepsilon subunits against the hexameric alpha(3)beta(3) subunits is highly coordinative, driven by ATP hydrolysis and structural changes at three beta subunits. However, the dynamical and coordinating structural transitions in the beta subunits are not fully understood at the molecular level. Here we examine structural transitions and domain motions in the active subunits of F1-ATPase via dynamical domain analysis of the alpha(3)beta(3)gammadeltaepsilon complex. The domain movement and hinge axes and bending residues have been identified and determined for various conformational changes of the beta-subunits. P-loop and the ATP-binding pocket are for the first time found to play essential mechanical functions additional to the catalytic roles. The cooperative conformational changes pertaining to the rotary mechanism of F1-ATPase appears to be more complex than Boyer's 'bi-site' activity. These findings provide unique molecular insights into dynamic and cooperative domain motions in F1-ATPase.     

pH dependence of hydrogen exchange from backbone peptide amides in apamin

The kinetics of hydrogen exchange of the 11 most protected backbone amides of bee venom apamin have been measured between pH 1 and pH 8.5 by using time-resolved and saturation-transfer NMR spectroscopy. The five amides most protected from base-catalyzed exchange, those of residues 5 and 12-15, show highly correlated exchange behavior in the base-catalyzed regime. It is proposed that the intramolecular hydrogen bonds stabilizing these amides define a stable cooperative unit of secondary structure in apamin (a C-terminal helix and an N-terminal beta-turn). This conformational unit is further stabilized (by 5-6 kJ mol-1) on titration of the Glu-7 side-chain carboxyl group. The relative contributions of specific intramolecular interactions to this conformational stabilization are estimated. The pHminima in the pH-dependent single amide exchange curves are compared with values predicted by correcting for sequence-dependent contributions to amide exchange rates [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. The lack of correlation suggests that the "open" conformers from which amide exchange occurs are nonrandom. This conclusion is dependent on the assumption that acid-catalyzed exchange occurs via N-protonation so that residual conformational effects on exchange rates in the open conformers will affect acid- and base-catalyzed rates in approximately equal and opposite ways. A strong correlation between the measured pHminima and the amide proton chemical shifts is observed, however, and this may be most easily accommodated if acid-catalyzed exchange occurs by the imidic acid mechanism (via amide O-protonation).     

A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding

The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.     

Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides

We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.     

The molecular mechanism of toxin-induced conformational changes in a potassium channel: relation to C-type inactivation

Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.     

Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.     

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.     

Cooperative transition in the conformation of 24-mer tarantula hemocyanin upon oxygen binding

Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula hemocyanin. Oxygen binding of the hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding curves recorded with tryptophan and Prodan emission revealed that structural changes in tarantula hemocyanin lag behind oxygen binding at the beginning of oxygenation. Analyses based on the nested two-state model, which describes cooperative oxygen binding of hemocyanins, indicated that the transition monitored by Prodan emission is closely related to one of the four conformations (rR) predicted for the allosteric unit. Earlier, the allosteric unit of tarantula hemocyanin was found to be the 12-mer half-molecule. Here, fluorescence titration revealed that the number of Prodan binding sites/24-mer tarantula hemocyanin is approximately 2, matching the number of allosteric units/hemocyanin. Based on the agreement between oxygen binding curves and fluorescence titration we concluded that Prodan monitors a conformational transition of the allosteric unit.     

Machinery of protein folding and unfolding

During the past two years, a large amount of biochemical, biophysical and low- to high-resolution structural data have provided mechanistic insights into the machinery of protein folding and unfolding. It has emerged that dual functionality in terms of folding and unfolding might exist for some systems. The majority of folding/unfolding machines adopt oligomeric ring structures in a cooperative fashion and utilise the conformational changes induced by ATP binding/hydrolysis for their specific functions.     

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.     

Allosterism and cooperativity in Pseudomonas aeruginosa GDP-mannose dehydrogenase

GDP-Mannose dehydrogenase catalyzes the formation of GDP-mannuronic acid, which is the monomeric unit from which the polysaccharide alginate is formed. Alginate is secreted by the pathogenic bacterium Pseudomonas aeruginosa and is believed to play an important role in the bacteria's resistance to antibiotics and the host immune response. We have characterized the kinetic behavior of GDP-mannose dehydrogenase in detail. The enzyme displays cooperative behavior with respect to NAD(+) binding, and phosphate and GMP act as allosteric effectors. Binding of the allosteric effectors causes the Hill coefficient for NAD(+) binding to decrease from 6 to 1, decreases K(1/2) for NAD(+) by a factor of 10, and decreases V(max) by a factor of 2. The cooperative binding of NAD(+) is also sensitive to pH; deprotonation of two residues with identical pK's of 8.0 is required for maximally cooperative behavior. The kinetic behavior of GDP-mannose dehydrogenase suggests that it must be at least hexameric under turnover conditions; however, dynamic light-scattering measurements do not provide a clear determination of the size of the active enzyme complex.     

SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.     

Effects of temperature and pH on the water exchange through erythrocyte membranes: Nuclear magnetic resonance studies

Summary The temperature and pH dependence of water exchange has been studied on isolated erythrocytes suspended in isotonic buffered solutions. At pH 7.4 a break in the Arrhenius plot of water exchange time at around 26°C was found. The mean value of the apparent activation energy of the water exchange time at temperatures higher than that of the discontinuity was 5.7 kcal/mole (±0.4); at lower temperatures the values of the apparent activation energy were below 1.4 kcal/mole. The pH dependence of water exchange time of isolated erythrocytes revealed a marked increase of the water exchange time values in the acid range of pH; a much smaller variation of the same parameter occurs between pH 7.0 and 8.0. These finding could be correlated with other processes involving erythrocyte membranes that showed similar pH and temperature dependence and were considered to indicate state transitions in the membranes. It is suggested that the temperature and pH effects on water diffusion indicate that conformational changes and cooperative effects are implicated in the mechanism of this transport process.Institute for Isotopic and Molecular Technology.     

Recessiveness and dominance in barley mutants deficient in Mg-chelatase subunit D, an AAA protein involved in chlorophyll biosynthesis

Mg-chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX at the first committed step of the chlorophyll biosynthetic pathway. It consists of three subunits: I, D, and H. The I subunit belongs to the AAA protein superfamily (ATPases associated with various cellular activities) that is known to form hexameric ring structures in an ATP-dependant fashion. Dominant mutations in the I subunit revealed that it functions in a cooperative manner. We demonstrated that the D subunit forms ATP-independent oligomeric structures and should also be classified as an AAA protein. Furthermore, we addressed the question of cooperativity of the D subunit with barley (Hordeum vulgare) mutant analyses. The recessive behavior in vivo was explained by the absence of mutant proteins in the barley cell. Analogous mutations in Rhodobacter capsulatus and the resulting D proteins were studied in vitro. Mixtures of wild-type and mutant R. capsulatus D subunits showed a lower activity compared with wild-type subunits alone. Thus, the mutant D subunits displayed dominant behavior in vitro, revealing cooperativity between the D subunits in the oligomeric state. We propose a model where the D oligomer forms a platform for the stepwise assembly of the I subunits. The cooperative behavior suggests that the D oligomer takes an active part in the conformational dynamics between the subunits of the enzyme.