Nucleotide-induced Conformational Changes Research Articles

Substituent Effects on Transient, Carbodiimide-Induced Geometry Changes in Diphenic Acids.

Nucleotide-induced conformational changes in motor proteins are key to many important cell functions. Inspired by this biological behavior, we report a simple chemically fueled system that exhibits carbodiimide-induced geometry changes. Bridging via transient anhydride formation leads to a significant reduction of the twist about the biaryl bond of substituted diphenic acids, giving a simple molecular clamp. The kinetics are well-described by a simple mechanism, allowing structure-property effects to be determined. The kinetic parameters can be used to derive important characteristics of the system such as the efficiencies (anhydride yields), maximum anhydride concentrations, and overall lifetimes. Transient diphenic anhydrides tolerate steric hindrance ortho to the biaryl bond but are significantly affected by electronic effects, with electron-deficient substituents giving lower yields, peak conversions, and lifetimes. The results provide useful guidelines for the design of functional systems incorporating diphenic acid units.

Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication.

Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase

RNA-dependent NTPases can act as RNA/RNA-protein remodeling enzymes and typically exhibit low NTPase activity in the absence of RNA/RNA-protein substrates. How futile intrinsic NTP hydrolysis is prevented is frequently not known. The ATPase/RNA helicase Brr2 belongs to the Ski2-like family of nucleic acid-dependent NTPases and is an integral component of the spliceosome. Comprehensive nucleotide binding and hydrolysis studies are not available for a member of the Ski2-like family. We present crystal structures of Chaetomium thermophilum Brr2 in the apo, ADP-bound, and ATPγS-bound states, revealing nucleotide-induced conformational changes and a hitherto unknown ATPγS binding mode. Our results in conjunction with Brr2 structures in other molecular contexts reveal multiple molecular mechanisms that contribute to the inhibition of intrinsic ATPase activity, including an N-terminal region that restrains the RecA-like domains in an open conformation and exclusion of an attacking water molecule, and suggest how RNA substrate binding can lead to ATPase stimulation.

Head-bent resistant Hsc70 variants show reduced Hsp40 affinity and altered protein folding activity

The molecular chaperone Hsc70 performs essential tasks by folding proteins. Hsc70 is driven by the hydrolysis of ATP and tuned by the association with various co-chaperones. One such cofactor is the nematode nucleotide exchange factor UNC-23, whose mutation disrupts muscle attachment and induces a severe head-bent phenotype in C.elegans. Interestingly, four mutations in Hsc70 can suppress this phenotype, but the molecular mechanism underlying this suppression is unknown. Here we characterize these four suppressor variants, Hsc70 D233N, S321F, A379V and D384N. In vitro only Hsc70 S321F shows reduced stability and altered nucleotide interaction, but all mutations affect the ATPase stimulation. In particular, Hsc70 D233N and Hsc70 A379V show strongly reduced interactions with DNJ-12 and DNJ-13. Nucleotide exchange factor binding instead is barely influenced in Hsc70 D233N, A379V and D384N and their chaperone activity is preserved. Molecular dynamics simulations suggest that effects in Hsc70 S321F and Hsc70 A379V originate from steric clashes in the vicinity of the mutation site, while D233N disrupts a salt bridge that contributes to Hsc70’s nucleotide-induced conformational changes. In summary, the analyzed mutants show altered ATPase and refolding activity caused by changes in Hsp40 binding.

The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1.

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

High concentrations of GTP induce conformational changes in the essential bacterial GTPase EngA and enhance its binding to the ribosome.

EngA is a conserved bacterial GTPase involved in ribosome biogenesis. While essential in bacteria, EngA does not have any human orthologue and can thus be an interesting target for new antibacterial compounds. EngA is the only known GTPase bearing two G domains, making unique its catalytic cycle and the induced modulation of its conformation and interaction with the ribosome. We have investigated nucleotide-induced conformational changes in EngA in order to unveil their role in ribosome binding. SAXS and limited proteolysis were used to probe EngA conformational changes, and revealed a change in protein structure and a distinct rate of proteolysis induced by GTP. Structure analysis showed that the conformation adopted in solution in the presence of GTP does not match any known EngA structure, while the SAXS data measured in the presence of GDP are in perfect agreement with two crystal structures (i.e. 2HGJ and 4DCU). Combination of mass spectrometry and N-terminal sequencing for the analysis of the fragmentation pattern upon proteolytic cleavage gave insights into which regions become more or less accessible in the different nucleotide-bound states. Interactions studies confirmed a stronger binding of EngA to the bacterial ribosome in the presence of GTP and suggest that the induced change in conformation of EngA plays a key role for ribosome binding.

Interaction between the AAA+ ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding

p97 is an essential ATPase associated with various cellular activities (AAA+) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum-associated degradation. However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants.

The Interplay of Cofactor Interactions and Post-translational Modifications in the Regulation of the AAA+ ATPase p97

The hexameric type II AAA ATPase (ATPase associated with various activities) p97 (also referred to as VCP, Cdc48, and Ter94) is critically involved in a variety of cellular activities including pathways such as DNA replication and repair which both involve chromatin remodeling, and is a key player in various protein quality control pathways mediated by the ubiquitin proteasome system as well as autophagy. Correspondingly, p97 has been linked to various pathophysiological states including cancer, neurodegeneration, and premature aging. p97 encompasses an N-terminal domain, two highly conserved ATPase domains and an unstructured C-terminal tail. This enzyme hydrolyzes ATP and utilizes the resulting energy to extract or disassemble protein targets modified with ubiquitin from stable protein assemblies, chromatin and membranes. p97 participates in highly diverse cellular processes and hence its activity is tightly controlled. This is achieved by multiple regulatory cofactors, which either associate with the N-terminal domain or interact with the extreme C-terminus via distinct binding elements and target p97 to specific cellular pathways, sometimes requiring the simultaneous association with more than one cofactor. Most cofactors are recruited to p97 through conserved binding motifs/domains and assist in substrate recognition or processing by providing additional molecular properties. A tight control of p97 cofactor specificity and diversity as well as the assembly of higher-order p97-cofactor complexes is accomplished by various regulatory mechanisms, which include bipartite binding, binding site competition, changes in oligomeric assemblies, and nucleotide-induced conformational changes. Furthermore, post-translational modifications (PTMs) like acetylation, palmitoylation, phosphorylation, SUMOylation, and ubiquitylation of p97 have been reported which further modulate its diverse molecular activities. In this review, we will describe the molecular basis of p97-cofactor specificity/diversity and will discuss how PTMs can modulate p97-cofactor interactions and affect the physiological and patho-physiological functions of p97.

Probing nucleotide-induced conformational changes and interaction studies of the GTPase EngA

Probing nucleotide-induced conformational changes and interaction studies of the GTPase EngA

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect single-stranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5-30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.

Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin.

DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

Lis1 restricts the conformational changes in cytoplasmic dynein on microtubules.

Cytoplasmic dynein is a microtubule-based motor protein that transports intracellular cargo and performs various functions during cell division. We previously reported that Lis1 suppressed dynein motility on microtubules in an idling state. Recently, a model showed that Lis1 prevents the ATPase domain of dynein from transmitting a detachment signal to its microtubule-binding domain. However, conformational information on dynein is limited. We used electron microscopy to investigate the conformation of dynein and nucleotide-induced conformational changes on microtubules. The conformation of dynein differed depending on the presence or absence of a nucleotide. In the presence of the nucleotide ADP-vanadate, dynein displayed an extended form on microtubules (extended form), whereas in the absence of a nucleotide, dynein lay along microtubules (compact form). This conformational change reflects chemomechanical coupling in dynein walking on microtubules. We also found that Lis1 fixed the conformation of dynein in the compact form regardless of the nucleotide condition. Removal of the Lis1 dimerization motif abolished Lis1-dependent fixation of dynein in the compact form. This suggests that the idling state of dynein on microtubules induced by Lis1 occurs through the Lis1-dependent arrest of dynein chemomechanical coupling.

Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ.

Y-family DNA polymerases are specialized to copy damaged DNA, and are associated with increased mutagenesis, owing to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer, but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have, to date, not been extensively characterized. Using hydrogen-deuterium exchange mass spectrometry, we demonstrate here that the Escherichiacoli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N(2) -furfuryl-deoxyguanosine, which is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O(6) -methyl-deoxyguanosine, which is a poor substrate. Our observations suggest that DinB and DNA polymerase κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

Non‐cognate DNA damage prevents formation of active conformation of Y‐family DNA polymerases DinB and pol kappa

Y‐family DNA polymerases are specialized to copy damaged DNA and are associated with an increased risk of mutagenesis due to their low fidelity. It is believed that the mechanism of nucleotide selection by Y‐family DNA polymerases involves conformational changes preceding nucleotidyl transfer but there is limited experimental evidence for such structural changes. Using hydrogen/deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y‐family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide‐induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2‐furfuryl‐deoxyguanosine that is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6‐methyl‐deoxyguanosine that is a poor substrate. Structural changes were localized to regions both near and far from the active site and involved several domains of the enzymes. Our observations suggest that DinB and pol κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.

Reprint of "The mechanism of negative DNA supercoiling: a cascade of DNA-induced conformational changes prepares gyrase for strand passage".

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling. Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.

The Acidic C-terminal Tail of the GyrA Subunit Moderates the DNA Supercoiling Activity of Bacillus subtilis Gyrase

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.

The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling. Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.

SAXS analysis of the tRNA-modifying enzyme complex MnmE/MnmG reveals a novel interaction mode and GTP-induced oligomerization.

Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle.

Nucleotide-induced conformational changes of tetradecameric GroEL mapped by H/D exchange monitored by FT-ICR mass spectrometry.

Here we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) to access E. coli chaperonin GroEL conformation. The ~800 kDa tetradecameric GroEL plays an essential role in the proper folding of many proteins. Previous studies of the structural dynamics of GroEL upon ATP binding have been inconsistent, showing either minimal or major allosteric changes. Our results, based on the native, non-mutated, protein under physiological conditions in solution demonstrate substantial changes in conformation and/or flexibility upon ATP binding. We capture the pivotal step in its functional cycle by use of a non-hydrolyzable ATP analog, ATPγS, to mimic the ATP-bound GroEL state. Comparison of HDX-MS results for apo GroEL and GroEL-ATPγS enables the characterization of the nucleotide-regulated conformational changes throughout the entire protein with high sequence resolution. The 14-mer GroEL complex is the largest protein assembly yet accessed by HDX-MS, with sequence resolution of segments of as few as five amino acids.

Miro: A Driver of the Kinesin Motor

Kinesin-1 is the primary anterograde microtubule-based motor for mitochondria. Kinesin-1 binds to a unique cargo-binding adaptor protein, Milton, and Milton attaches to the outer mitochondrial membrane protein Miro, tethering kinesin-1 to mitochondria. The distribution of mitochondria within neurons is particularly important. Defects in this transport may underlie several neurodegenerative diseases such as Parkinson's disease. Recent work has identified Miro as a calcium (Ca) dependent regulator of kinesin-1-mediated mitochondrial motility. Miro contains two GTPase domains that flank two Ca-binding EF-hand domains. In this work, we show that purified Miro protein binds both Ca and guanine nucleotides in vitro, but not adenine nucleotides. We also report that Miro does not undergo large conformational changes upon binding Ca or guanine nucleotides, as measured by small angle x-ray scattering. These findings suggest that the presence of a protein binding partner may be required for Ca- and/or nucleotide-induced conformational changes.