Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis,Neurodegeneration, and Impaired Behavior

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.A variety of both exogenous and endogenous reactive compounds present a constant threat to the integrity of DNA in living cells. DNA damage introduced by such compounds can lead to high and deleterious mutation rates as well as DNA cytotoxicity, both to the nuclear and the mitochondrial genome. This has triggered the evolution of several different DNA repair pathways (28). One is the base excision repair (BER) pathway, which repairs small base alterations that do not distort the DNA helix. Repair of such highly abundant lesions by BER is performed by a multistep process that is initiated by a damage-specific DNA glycosylase, which removes the damaged base. One of these glycosylases is uracil-DNA glycosylase (UDG), which acts to preserve the genome by removing mutagenic uracil residues from the DNA. This glycosylase, as well as the OGG1 glycosylase that is specialized for the removal of oxidized bases, exists in a nuclear and mitochondrial splice form (1, 11, 37, 45). Accordingly, BER of a variety of lesions has been observed in mitochondria (26, 31).Damage to the mitochondrial DNA (mtDNA) can cause respiratory chain deficiency and lead to disorders that have varied phenotypes (35, 41). Many involve neurological features that are often associated with cell loss within specific brain regions. These pathologies, along with the increasing evidence of a decline in mitochondrial function with aging, have raised speculation that key changes in mitochondrial DNA sequences and functions could have a vital role in age-related neurodegenerative diseases (41). This has also been studied in several model organisms. Mouse models with respiratory chain deficient dopamine neurons have demonstrated adult onset Parkinsonism phenotype (16), and cell death induced by mitochondrial toxicity is likely to underlie Alzheimer disease (32). Mitochondrial oxidative stress and accumulation of mtDNA damage are believed to be particularly devastating to postmitotic differentiated tissue, including neurons (30). The mtDNA contains genetic information for 13 polypeptides that are a part of the electron transport chain and for rRNAs and tRNAs that are necessary for mitochondrial protein synthesis. Thus, damage to the mtDNA genome will affect the energetic capacities of the mitochondria and also influence the level of reactive oxygen species (ROS) and ultimately the susceptibility to apoptosis (30, 35).Some recent influential studies have assessed the effect of mtDNA mutagenesis, including small base-pair substitutions and larger mtDNA deletions, on the life span of mice. It was concluded that a massive increase in the frequency of mtDNA base-pair substitutions are required for inducing premature aging, whereas the number of mtDNA deletions coincides better with natural aging (25, 47-49).In the present study, we have combined two novel transgenic mouse models, which allow the induction of a high number of apyrimidinic (AP) sites specifically to the mitochondrial genome in adults simply by the addition of doxycycline to the diet. Such AP sites are created by the expression of a mutated version of mitochondrion-targeted human UDG (abbreviated here as mutUNG1), whereby an amino acid substitution results in an enzyme that removes thymine, in addition to uracil, from DNA (23). The CaMKIIα promoter restricts expression of the mutUNG1 to forebrain neurons (34). We demonstrate that a continuous generation of AP sites leads to apoptosis, accelerated neurodegeneration, and impaired behavior.     

A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.     

A template RNA entry channel in the fingers domain of the poliovirus polymerase

Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase, 3D(pol), that is required for viral RNA replication. Structures of 3D(pol) from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a series of alanine substitution mutations at these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex (EC) stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding, while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for EC stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3D(pol) pinky finger drastically decreased the rate of initiation as well as EC stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or EC stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA ECs.     

Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.Members of the Picornaviridae family of small RNA viruses cause a wide range of diseases in humans, including liver disease, heart disease, aseptic meningitis, the common cold, and poliomyelitis. The picornaviruses include the most common human viruses, which are the rhinoviruses that spread through respiratory pathways, and the second most common viruses, which are enteroviruses that spread by fecal-oral transmission. These viruses have ≈7.5-kb positive-sense genomes containing a single large open reading frame encoding a ≈250-kDa polyprotein that is cleaved into about a dozen different proteins by viral proteases (20). Their genome life cycle is completely RNA based, with replication being driven by the viral 3D^pol protein, an RNA-dependent RNA polymerase (RdRP).After viral RNA translation and polyprotein processing, 3D^pol replicates the infecting positive-strand RNA template into a negative-strand intermediate that is subsequently used as a template for positive-strand synthesis. During these processes, 3D^pol interacts with multiple templates, substrates, and other viral proteins; however, many aspects of these events remain obscure. The crystal structures of several picornaviral 3D^pol enzymes have been solved, and these all conform to the “right hand” analogy commonly used to describe polymerases as having palm, thumb, and finger domains (10, 14, 18, 22, 27, 29). Based on homology to other polymerases and the structures of 3D^pol-RNA complexes with foot-and-mouth disease and Norwalk virus polymerases, the finger domain interacts with the template RNA, the palm domain contains the active-site aspartate residues that coordinate the metals needed for catalysis, and the thumb domain contacts the exiting duplex RNA product (13, 26, 30).Poliovirus is among the most-studied picornaviruses, and its polymerase has been thoroughly characterized biochemically (3, 15) and structurally (28, 29). Processive RNA synthesis requires the formation of a stable 3D^pol elongation complex through a multistep process involving at least two conformational changes (2). First, following RNA binding, there is a slow (t_1/2 ≈ 12 s) conformational change that results in a 3D^pol-RNA complex poised for nucleoside triphosphate (NTP) incorporation. Second, following the addition of the first nucleotide to the primer, there is another conformational change to produce a very stable elongation complex with an in vitro half-life on the order of several hours. The polymerase begins processive elongation after the formation of this stable elongation complex, and each nucleotide addition cycle involves a five-step mechanism, of which NTP repositioning and NTP catalysis are rate limiting (3). Similar experiments using the homologous foot-and-mouth disease virus polymerase reveal an analogous set of complexes resulting in an elongation complex with a half-life of 27 h (1). Although these viral polymerase complexes have been well characterized biochemically, there is relatively little known about any structural changes involved in elongation complex formation or the catalytic cycle itself; all the 3D^pol structures solved thus far show essentially the same conformation, with no evidence for significant conformational changes upon RNA or NTP binding.The activation of several picornaviral polymerases is dependent upon correct cleavage of 3D^pol from the viral 3CD^pro precursor protein in order to create a new N terminus that can be buried in a pocket at the base of the 3D^pol finger domain. This buried N terminus has been observed in poliovirus, coxsackievirus, rhinovirus, and foot-and-mouth disease virus polymerases (10, 14, 22, 29). In solving the structure of poliovirus polymerase, we observed that burying the N terminus resulted in a subtle but important conformational change in the active site whereby Asp238 was repositioned to make a key hydrogen bond with the 2′ hydroxyl of a bound NTP (29). Addition or deletion of a single residue at the N terminus abolished enzyme activity, and mutation of Gly1 to alanine resulted in a partially active enzyme with slightly altered positioning of Asp238. Further data from coxsackievirus polymerase showed that the addition of a second N-terminal glycine also inactivated the enzyme, but activity could be restored by also deleting Glu2, indicating that there is a specific length requirement in the N-terminal sequence of the enzyme (10). A prime candidate for involvement in such a length requirement is residue Phe5 of coxsackievirus 3D^pol that corresponds to Trp5 in poliovirus 3D^pol. In the 3D^pol structures, there is a backbone distortion in the β-strand composed of residues 1 to 9 that results in this large hydrophobic amino acid being solvent exposed rather than buried in an adjacent hydrophobic pocket (Fig. ​(Fig.11 A). This unusual conformation at residue 5 is conserved among picornaviral polymerase structures, and substitution mutations at this residue had significant effects on coxsackievirus polymerase activity (10). Large hydrophobic amino acids at residue 5 increased 3D^pol activity, while small amino acids at residue 5 decreased 3D^pol activity (10). Based on these data, we previously proposed that the 3D^pol catalytic cycle involves a conformational change wherein residue 5 flips into an adjacent hydrophobic patch on the polymerase to aid in NTP positioning, and such a conformational change would require the N terminus to be correctly buried to act as a stable pivot for the rotational movement.Open in a separate windowFIG. 1.Elongation complex formation. (A) Structure of poliovirus polymerase showing the distortion of the β-sheet conformation between residues 3 and 4 that results in Trp5 being solvent exposed adjacent to a large hydrophobic patch composed of residues from the index (green) and middle (orange) fingers. (B) Cartoon of the PETE (polymerase elongation template element) RNAs used in complex formation assays. Both RNAs are G-less until the sixth nucleotide from the end, which limits secondary structure and stops elongation before the 5′ end to avoid possible end effects. The asterisk indicates the position of the amino-modified deoxythymidine where the IRDye label is covalently attached. (C) Denaturing PAGE showing the time course for formation of the +1 and +2 products from the two RNAs. Elongation complexes were formed by incubating 1 μM each PETE RNA, 15 μM 3D^pol, and 40 μM ATP and GTP at 22°C for various times as indicated. (D) Kinetics of +2 complex formation rates obtained from band intensity data curve fit to a single exponential. The resulting formation time constants are listed in Table ​Table11.In this work, we have investigated the role of residue 5 in 3D^pol in further detail by examining how a series of mutations in poliovirus 3D^pol affects RNA binding, elongation complex formation, elongation rate, and elongation complex stability. The data show that residue 5 mutations have major effects on the stability of the elongation complex, with minor effects on elongation complex formation and no effect on RNA binding affinities and elongation rates. Replication defects are also observed in the context of viral replication centers where residue 5 mutations significantly reduce RNA synthesis in cell-free coupled translation-replication reactions and slow the growth of infectious virus in cells.     

Estimating Extracellular Spike Waveforms from CA1 Pyramidal Cells with Multichannel Electrodes

Extracellular (EC) recordings of action potentials from the intact brain are embedded in background voltage fluctuations known as the “local field potential” (LFP). In order to use EC spike recordings for studying biophysical properties of neurons, the spike waveforms must be separated from the LFP. Linear low-pass and high-pass filters are usually insufficient to separate spike waveforms from LFP, because they have overlapping frequency bands. Broad-band recordings of LFP and spikes were obtained with a 16-channel laminar electrode array (silicone probe). We developed an algorithm whereby local LFP signals from spike-containing channel were modeled using locally weighted polynomial regression analysis of adjoining channels without spikes. The modeled LFP signal was subtracted from the recording to estimate the embedded spike waveforms. We tested the method both on defined spike waveforms added to LFP recordings, and on in vivo-recorded extracellular spikes from hippocampal CA1 pyramidal cells in anaesthetized mice. We show that the algorithm can correctly extract the spike waveforms embedded in the LFP. In contrast, traditional high-pass filters failed to recover correct spike shapes, albeit produceing smaller standard errors. We found that high-pass RC or 2-pole Butterworth filters with cut-off frequencies below 12.5 Hz, are required to retrieve waveforms comparable to our method. The method was also compared to spike-triggered averages of the broad-band signal, and yielded waveforms with smaller standard errors and less distortion before and after the spike.     

Distinct Conformations of a Putative Translocation Element in Poliovirus Polymerase

The mechanism whereby RNA is translocated by the single subunit viral RNA-dependent RNA polymerases is not yet understood. These enzymes lack homologs of the “O-helix” structures and associated fingers domain movements thought to be responsible for translocation in many DNA-templated polymerases. The structures of multiple picornavirus polymerase elongation complexes suggest that these enzymes use a different molecular mechanism where translocation is not strongly coupled to the opening of the active site following catalysis. Here we present the 2.0- to 2.6-Å-resolution crystal structures and biochemical data for 12 poliovirus polymerase mutants that together show how proper enzyme functions and translocation activity requires conformational flexibility of a loop sequence in the palm domain B-motif. Within the loop, the Ser288-Gly289-Cys290 sequence is shown to play a major role in the catalytic cycle based on RNA binding, processive elongation activity, and single nucleotide incorporation assays. The structures show that Ser288 forms a key hydrogen bond with Asp238, the backbone flexibility of Gly289 is required for translocation competency, and Cys290 modulates the overall elongation activity of the enzyme. Some conformations of the loop represent likely intermediates on the way to forming the catalytically competent closed active site, while others are consistent with a role in promoting translocation of the nascent base pair out of the active site. The loop structure and key residues surrounding it are highly conserved, suggesting that the structural dynamics we observe in poliovirus 3D^pol are a common feature of viral RNA-dependent RNA polymerases.     

Dimeric structure of the Oxytricha nova telomere end-binding protein alpha-subunit bound to ssDNA

Telomeres are the specialized protein--DNA complexes that cap and protect the ends of linear eukaryotic chromosomes. The extreme 3' end of the telomeric DNA in Oxytricha nova is bound by a two-subunit sequence-specific and 3' end-specific protein called the telomere end-binding protein (OnTEBP). Here we describe the crystal structure of the alpha-subunit of OnTEBP in complex with T4G4 single-stranded telomeric DNA. This structure shows an (alpha--ssDNA)2 homodimer with a large approximately 7,000 A2 protein--protein interface in which the domains of alpha are rearranged extensively from their positions in the structure of an alpha--beta--ssDNA ternary complex. The (alpha--ssDNA)2 complex can bind two telomeres on opposite sides of the dimer and, thus, acts as a protein mediator of telomere--telomere associations. The structures of the (alpha--ssDNA)2 dimer presented here and the previously described alpha--beta--ssDNA complex demonstrate that OnTEBP forms multiple telomeric complexes that potentially mediate the assembly and disassembly of higher order telomeric structures.     

Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer

Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.