How directional translocation is regulated in a DNA helicase motor

PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally known in full atomic detail. It translocates progressively from the 3' end to the 5' end of single-stranded DNA utilizing the free energy from ATP hydrolysis. The similarities in structure and reaction pathway between PcrA helicase and F1-ATPase suggest a similar mechanochemical mechanism at work in both systems. Previous studies of PcrA translocation demonstrated a domain stepping mechanism in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of two translocation domains is synchronized with alternating mobilities of the individual domains such that PcrA moves unidirectionally along single-stranded DNA. To substantiate this translocation mechanism, this study applies molecular dynamics simulations, elastic network theory, and multiple sequence alignment to analyze the system. The analysis provides further evidence that directional translocation of PcrA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins.     

Insights into the DNA cleavage mechanism of human LINE-1 retrotransposon endonuclease

The human LINE-1 endonuclease (L1-EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1-EN could help engineering them as genetic tools. We tested the in vitro activity of point mutants in three L1-EN residues--Asp145, Arg155, Ile204--that are key for DNA cleavage, and determined their crystal structures. The L1-EN structure remains overall unaffected by the mutations, which change the enzyme activity but leave DNA cleavage sequence specificity mostly unaffected. To better understand the mechanism of L1-EN, we performed molecular dynamics simulations using as model the structures of wild type EN-L1, of two betaB6-betaB5 loop exchange mutants we have described previously to be important for DNA recognition, of the R155A mutant from this study, and of the homologous TRAS1 endonuclease: all confirm a rigid scaffold. The simulations crucially indicate that the betaB6-betaB5 loop shows an anticorrelated motion with the surface loops betaA6-betaA5 and betaB3-alphaB1. The latter loop harbors N118, a residue that alters DNA cleavage specificity in homologous endonucleases, and implies that the plasticity and correlated motion of these loops has a functional importance in DNA recognition and binding. To further explore how these loops are possibly involved in DNA binding, we docked computationally two DNA substrates to our structure, one involving a flipped-out nucleotide downstream the scissile phosphodiester; and one not. The models for both scenarios are feasible and agree with the hypotheses derived from the dynamic simulations. The reduced cleavage activity we have observed for the I204Y mutant above however, favors the flipped out nucleotide model.     

PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis , in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus . This gene is located at 55° on the chromosome and belongs to a putative operon together with a ligase gene ( lig ) and two unknown genes named pcrB and yerH . As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli . PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep⁻ phenotype on E. coli . Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.     

Increased flexibility as a strategy for cold adaptation: a comparative molecular dynamics study of cold- and warm-active uracil DNA glycosylase

Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway and removes uracil from the DNA strand. Atlantic cod UDG (cUDG), which is a cold-adapted enzyme, has been found to be up to 10 times more catalytically active in the temperature range 15-37 degrees C as compared with the warm-active human counterpart. The increased catalytic activity of cold-adapted enzymes as compared with their mesophilic homologues are partly believed to be caused by an increase in the structural flexibility. However, no direct experimental evidence supports the proposal of increased flexibility of cold-adapted enzymes. We have used molecular dynamics simulations to gain insight into the structural flexibility of UDG. The results from these simulations show that an important loop involved in DNA recognition (the Leu(272) loop) is the most flexible part of the cUDG structure and that the human counterpart has much lower flexibility in the Leu(272) loop. The flexibility in this loop correlates well with the experimental k(cat)/K(m) values. Thus, the data presented here add strong support to the idea that flexibility plays a central role in adaptation to cold environments.     

Molecular dynamics studies on the domain swapped Salmonella typhimurium survival protein SurE: insights on the possible reasons for catalytic cooperativity

Stationary phase survival protein SurE from Salmonella typhimurium is a dimeric protein formed by the swapping of a tetramerization loop involved in the formation of a loose tetramer and a C-terminal helix. It functions as a phosphatase. The two-fold symmetry of the dimeric protein was lost in the mutants H234A and D230A/H234A in which a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was eliminated. The catalytic activity of both mutants was drastically reduced. In contrast to the native protein, H234A exhibited positive cooperativity in its catalytic activity. In order to relate these observations to the dynamics of the native and distorted mutants, molecular dynamics (MD) simulations were carried out using GROMACS v4.0.7. In all the simulations, the swapped segments and a segment near the active site were found to be highly flexible. These segments exhibited distinct dynamic features in the two protomers (A and B) of the dimeric protein. The dimeric organization was more significantly affected in the mutants when compared to the native structure, suggesting that the mutations enhance the intrinsic flexibility of the protein. The larger flexibility of the mutants affects the relative movement between the loops near the two active sites. The positive cooperativity observed in H234A mutant is most likely due to this increased flexibility and loop movement.     

DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange

PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.     

Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non-hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single-stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an 'active' mechanism for PcrA that involves two distinct ATP-dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.     

Effect of DNA structural flexibility on promoter strength--molecular dynamics studies of E. coli promoter sequences

To study possible correlations between promoter activity and the structural flexibility of the DNA helix, we have carried out unrestrained molecular dynamics simulations of the -10 consensus region sequence and five variants forming the -10 region of various Escherichia coli promoter sequences. Analyses of the trajectories obtained from the simulations show that the consensus sequence has a pattern of two structurally flexible nucleotide steps sandwiched between two stiff steps. In the other sequences, this pattern varies in consonance with the change in the sequence. The variations in the patterns show correlation with the promoter strength.     

Brownian and essential dynamics studies of the HIV-1 integrase catalytic domain

The three-dimensional structure of the active site region of the enzyme HIV-1 integrase is not unambiguously known. This region includes a flexible peptide loop that cannot be well resolved in crystallographic determinations. Here we present two different computational approaches with different levels of resolution and on different time-scales to understand this flexibility and to analyze the dynamics of this part of the protein. We have used molecular dynamics simulations with an atomic model to simulate the region in a realistic and reliable way for 1 ns. It is found that parts of the loop wind up after 300 ps to extend an existing helix. This indicates that the helix is longer than in the earlier crystal structures that were used as basis for this study. Very recent crystal data confirms this finding, underlining the predictive value of accurate MD simulations. Essential dynamics analysis of the MD trajectory yields an anharmonic motion of this loop. We have supplemented the MD data with a much lower resolution Brownian dynamics simulation of 600 ns length. It provides ideas about the slow-motion dynamics of the loop. It is found that the loop explores a conformational space much larger than in the MD trajectory, leading to a "gating"-like motion with respect to the active site.     

Insights from xanthine and uracil DNA glycosylase activities of bacterial and human SMUG1: switching SMUG1 to UDG

Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only UDGs but also xanthine DNA glycosylases (XDGs). In addition, mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2 for XDG and UDG activities. Mutations at M57 (M57L) and H210 (H210G, H210M, and H210N), both of which are involved in interactions with the C2 carbonyl oxygen in uracil or xanthine, cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1, which corresponds to a position involved in base flipping. This mutation results in an activity profile resembling a human SMUG1-like enzyme as exemplified by the retention of UDG activity on mismatched base pairs and weak XDG activity. MD simulations indicate that M57L increases the flexibility of the motif 2 loop region and specifically A214, which may account for the reduced catalytic activity. G60Y completely abolishes XDG and UDG activity, which is consistent with a modeled structure in which G60Y blocks the entry of either xanthine or uracil to the base binding pocket. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive UDG as demonstrated by the uniform excision of uracil in both double-stranded and single-stranded DNA and the complete loss of XDG activity. MD simulations indicate that a combination of a reduced free volume and altered flexibility in the active-site loops may underlie the dramatic effects of the G63P mutation on the activity profile of SMUG1. This study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency.     

The study of interactions between DNA and PcrA DNA helicase by using targeted molecular dynamic simulations

DNA helicases are important enzymes involved in all aspects of nucleic acid metabolism, ranging from DNA replication and repair to recombination, rescue of stalled replication and translation. DNA helicases are molecular motors. Through conformational changes caused by ATP hydrolysis and binding, they move along the template double helix, break the hydrogen bonds between the two strands and separate the template chains, so that the genetic information can be accessed. In this paper, targeted molecular dynamic simulations were performed to study the important interactions between DNA and PcrA DNA helicase, which can not be observed from the crystal structures. The key residues on PcrA DNA helicase that have strong interactions with both double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) have been identified, and it was found that such interactions mostly exist between the protein and DNA backbone, which indicates that the translocation of PcrA is independent of the DNA sequence. The simulations indicate that the ds-DNA is separated upon ATP rebinding, rather than ATP hydrolysis, which suggests that the two strokes in the mechanism have two different major roles. Firstly, in the power stroke (ATP hydrolysis), most of the translocations of the bases from one pocket to the next occur. In the relaxation stroke (ATP binding), most of the ‘work’ is being done to ‘melt’ the DNA at the separation fork. Therefore, we propose a mechanism whereby the translocation of the ss-DNA is powered by ATP hydrolysis and the separation of the ds-DNA is powered by ATP binding.     

Relative flexibility of DNA and RNA: a molecular dynamics study

State of the art molecular dynamics simulations are used to study the structure, dynamics, molecular interaction properties and flexibility of DNA and RNA duplexes in aqueous solution. Special attention is paid to the deformability of both types of structures, revisiting concepts on the relative flexibility of DNA and RNA duplexes. Our simulations strongly suggest that the concepts of flexibility, rigidity and deformability are much more complex than usually believed, and that it is not always true that DNA is more flexible than RNA.     

Brownian and Essential Dynamics Studies of the HIV-1 Integrase Catalytic Domain

Abstract

The three-dimensional structure of the active site region of the enzyme HIV-1 integrase is not unambiguously known. This region includes a flexible peptide loop that cannot be well resolved in crystallographic determinations. Here we present two different computional approaches with different levels of resolution and on different time-scales to understand this flexibility and to analyze the dynamics of this part of the protein. We have used molecular dynamics simulations with an atomic model to simulate the region in a realistic and reliable way for 1 ns. It is found that parts of the loop wind up after 300 ps to extend an existing helix. This indicates that the helix is longer than in the earlier crystal structures that were used as basis for this study. Very recent crystal data confirms this finding, underlining the predictive value of accurate MD simulations. Essential dynamics analysis of the MD trajectory yields an anharmonic motion of this loop. We have supplemented the MD data with a much lower resolution Brownian dynamics simulation of 600 ns length. It provides ideas about the slow-motion dynamics of the loop. It is found that the loop explores a conformational space much larger than in the MD trajectory, leading to a “gating”-like motion with respect to the active site.     

The loop opening/closing motion of the enzyme triosephosphate isomerase.

To explore the origin of the large-scale motion of triosephosphate isomerase's flexible loop (residues 166 to 176) at the active site, several simulation protocols are employed both for the free enzyme in vacuo and for the free enzyme with some solvent modeling: high-temperature Langevin dynamics simulations, sampling by a "dynamics driver" approach, and potential-energy surface calculations. Our focus is on obtaining the energy barrier to the enzyme's motion and establishing the nature of the loop movement. Previous calculations did not determine this energy barrier and the effect of solvent on the barrier. High-temperature molecular dynamics simulations and crystallographic studies have suggested a rigid-body motion with two hinges located at both ends of the loop; Brownian dynamics simulations at room temperature pointed to a very flexible behavior. The present simulations and analyses reveal that although solute/solvent hydrogen bonds play a crucial role in lowering the energy along the pathway, there still remains a high activation barrier. This finding clearly indicates that, if the loop opens and closes in the absence of a substrate at standard conditions (e.g., room temperature, appropriate concentration of isomerase), the time scale for transition is not in the nanosecond but rather the microsecond range. Our results also indicate that in the context of spontaneous opening in the free enzyme, the motion is of rigid-body type and that the specific interaction between residues Ala176 and Tyr208 plays a crucial role in the loop opening/closing mechanism.     

Flexibility of prolyl oligopeptidase: molecular dynamics and molecular framework analysis of the potential substrate pathways

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.     

DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.     

Studies on somatostatin with time-resolved spectroscopy and molecular dynamics simulations

Somatostatin was studied by time-resolved fluorescence spectroscopy and by molecular dynamics simulations. The results obtained indicate the existence of mainly one conformation in DMSO, and the existence of several conformations in other solvents. Molecular dynamics simulations showed that the most important region for activity (residue 7-9) is also the most flexible region in the peptide.     

The Natively Disordered Loop of Bcl-2 Undergoes Phosphorylation-Dependent Conformational Change and Interacts with Pin1

Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2.