Wild-type Complex Research Articles

Principal component analysis of binding energies for single-point mutants of hT2R16 bound to an agonist correlate with experimental mutant cell response.

Directed evolution is a technique that enables the identification of mutants of a particular protein that carry a desired property by successive rounds of random mutagenesis, screening, and selection. This technique has many applications, including the development of G protein-coupled receptor-based biosensors and designer drugs for personalized medicine. Although effective, directed evolution is not without challenges and can greatly benefit from the development of computational techniques to predict the functional outcome of single-point amino acid substitutions. In this article, we describe a molecular dynamics-based approach to predict the effects of single amino acid substitutions on agonist binding (salicin) to a human bitter taste receptor (hT2R16). An experimentally determined functional map of single-point amino acid substitutions was used to validate the whole-protein molecular dynamics-based predictive functions. Molecular docking was used to construct a wild-type agonist-receptor complex, providing a starting structure for single-point substitution simulations. The effects of each single amino acid substitution in the functional response of the receptor to its agonist were estimated using three binding energy schemes with increasing inclusion of solvation effects. We show that molecular docking combined with molecular mechanics simulations of single-point mutants of the agonist-receptor complex accurately predicts the functional outcome of single amino acid substitutions in a human bitter taste receptor.

Functional insight into the role of Orc6 in septin complex filament formation in Drosophila.

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

GMXPBSA 2.1: A GROMACS tool to perform MM/PBSA and computational alanine scanning

GMXPBSA 2.1: A GROMACS tool to perform MM/PBSA and computational alanine scanning

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

N-terminal acetylation (Nt-acetylation) occurs on the majority of eukaryotic proteins and is catalyzed by N-terminal acetyltransferases (NATs). Nt-acetylation is increasingly recognized as a vital modification with functional implications ranging from protein degradation to protein localization. Although early genetic studies in yeast demonstrated that NAT-deletion strains displayed a variety of phenotypes, only recently, the first human genetic disorder caused by a mutation in a NAT gene was reported; boys diagnosed with the X-linked Ogden syndrome harbor a p.Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of the NatA complex, and suffer from global developmental delays and lethality during infancy. Here, we describe a Saccharomyces cerevisiae model developed by introducing the human wild-type or mutant NatA complex into yeast lacking NatA (NatA-Δ). The wild-type human NatA complex phenotypically complemented the NatA-Δ strain, whereas only a partial rescue was observed for the Ogden mutant NatA complex suggesting that hNaa10 S37P is only partially functional in vivo. Immunoprecipitation experiments revealed a reduced subunit complexation for the mutant hNatA S37P next to a reduced in vitro catalytic activity. We performed quantitative Nt-acetylome analyses on a control yeast strain (yNatA), a yeast NatA deletion strain (yNatA-Δ), a yeast NatA deletion strain expressing wild-type human NatA (hNatA), and a yeast NatA deletion strain expressing mutant human NatA (hNatA S37P). Interestingly, a generally reduced degree of Nt-acetylation was observed among a large group of NatA substrates in the yeast expressing mutant hNatA as compared with yeast expressing wild-type hNatA. Combined, these data provide strong support for the functional impairment of hNaa10 S37P in vivo and suggest that reduced Nt-acetylation of one or more target substrates contributes to the pathogenesis of the Ogden syndrome. Comparative analysis between human and yeast NatA also provided new insights into the co-evolution of the NatA complexes and their substrates. For instance, (Met-)Ala- N termini are more prevalent in the human proteome as compared with the yeast proteome, and hNatA displays a preference toward these N termini as compared with yNatA.

Functional characterization of the small regulatory subunit PetP from the cytochrome b6f complex in Thermosynechococcus elongatus.

The cyanobacterial cytochrome b(6)f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b(6)f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700(+) reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b(6)f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b(6)f complexes with different functions that might be correlated with supercomplex formation.

A multiscale approach to predicting affinity changes in protein-protein interfaces.

Substitution mutations in protein-protein interfaces can have a substantial effect on binding, which has consequences in basic and applied biomedical research. Experimental expression, purification, and affinity determination of protein complexes is an expensive and time-consuming means of evaluating the effect of mutations, making a fast and accurate in silico method highly desirable. When the structure of the wild-type complex is known, it is possible to economically evaluate the effect of point mutations with knowledge based potentials, which do not model backbone flexibility, but these have been validated only for single mutants. Substitution mutations tend to induce local conformational rearrangements only. Accordingly, ZEMu (Zone Equilibration of Mutants) flexibilizes only a small region around the site of mutation, then computes its dynamics under a physics-based force field. We validate with 1254 experimental mutants (with 1-15 simultaneous substitutions) in a wide variety of different protein environments (65 protein complexes), and obtain a significant improvement in the accuracy of predicted ΔΔG.

GMXPBSA 2.0: A GROMACS tool to perform MM/PBSA and computational alanine scanning

GMXPBSA 2.0 is a user-friendly suite of Bash/Perl scripts for streamlining MM/PBSA calculations on structural ensembles derived from GROMACS trajectories, to automatically calculate binding free energies for protein–protein or ligand–protein complexes. GMXPBSA 2.0 is flexible and can easily be customized to specific needs. Additionally, it performs computational alanine scanning (CAS) to study the effects of ligand and/or receptor alanine mutations on the free energy of binding. Calculations require only for protein–protein or protein–ligand MD simulations. GMXPBSA 2.0 performs different comparative analysis, including a posteriori generation of alanine mutants of the wild-type complex, calculation of the binding free energy values of the mutant complexes and comparison of the results with the wild-type system. Moreover, it compares the binding free energy of different complexes trajectories, allowing the study the effects of non-alanine mutations, post-translational modifications or unnatural amino acids on the binding free energy of the system under investigation. Finally, it can calculate and rank relative affinity to the same receptor utilizing MD simulations of proteins in complex with different ligands. In order to dissect the different MM/PBSA energy contributions, including molecular mechanic (MM), electrostatic contribution to solvation (PB) and nonpolar contribution to solvation (SA), the tool combines two freely available programs: the MD simulations software GROMACS and the Poisson–Boltzmann equation solver APBS. All the calculations can be performed in single or distributed automatic fashion on a cluster facility in order to increase the calculation by dividing frames across the available processors. The program is freely available under the GPL license. Program summaryProgram title: GMXPBSA 2.0Catalogue identifier: AETQ_v1_0Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AETQ_v1_0.htmlProgram obtainable from: CPC Program Library, Queen’s University, Belfast, N. IrelandLicensing provisions: GNU General Public License, version 3No. of lines in distributed program, including test data, etc.: 185937No. of bytes in distributed program, including test data, etc.: 7074217Distribution format: tar.gzProgramming language: Bash, Perl.Computer: Any computer.Operating system: Linux, Unix OS.RAM: ∼2 GBClassification: 3.External routines: APBS (http://www.Poissonboltzmann.org/apbs/) and GROMACS installations (http://www.gromacs.org). Optionally LaTeX.Nature of problem:Calculates the Molecular Mechanics (MM) data (Lennard-Jones and Coulomb terms) and the solvation energy terms (polar and nonpolar terms respectively) from an ensemble of structures derived from GROMACS molecular dynamics simulation trajectory. These calculations are performed for each single component of the simulated complex, including protein and ligand. In order to cancel out artefacts an identical grid setup for each component, including complex, protein and ligand, is required. Performs statistical analysis on the extracted data and comparison with wild-type complex in case of either computational alanine scanning or calculations on a set of simulations. Evaluates possible outliers in the frames extracted from the simulations during the binding free energy calculations.Solution method:The tool combines the freely available programs GROMACS and APBS to: 1.extract frames from a single or multiple complex molecular dynamics (MD) simulation, allowing comparison between multiple trajectories;2.split the complex frames in the single components including complex, protein and ligand;3.calculate the Lennard-Jones and Coulomb energy values (MM terms);4.calculate the polar solvation energy values using the implicit solvation Poisson–Boltzmann model (PB);5.calculate the nonpolar solvation energy value based on the solvent accessible surface area (SASA);6.combine all the calculated terms into the final binding free energy value;7.repeat the same procedure from point 1 to 6 for each simulation in case of computational alanine scanning (CAS) or simultaneous comparison of different MDs.Restrictions:Input format files compatible with GROMACS engine 4.5 and later versions. Availability of the force field or of the topology files.Running time:On a single core, Lennard-Jones, Coulomb and nonpolar solvation terms calculations require a few minutes. The time required for polar solvation terms calculations depends on the system size.

A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAyP2A complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9–16nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10–12nm comparable to the size of single TatAyP2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAyP2A complexes and we propose that an individual TatAyP2A complex assembles initially with a TatAyP2ACy complex, after which further TatAyP2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.

Site-directed mutagenesis of evolutionarily conserved serine residues in the N-terminal domain of rice Phi-class glutathione S-transferase F5

Abstract To identify the critical residue for the catalytic activity of rice glutathione S -transferases (GSTs), the evolutionarily conserved serine residues in the N-terminal domain of rice phi class GST F5 ( Os GST F5) were replaced with Ala by site-directed mutagenesis. Replacement of Ser9 with Ala significantly affected the catalytic activity and kinetic parameters of the enzyme, whereas substitutions of Ser34 and Ser65 with Ala showed little effect. The S9A mutant showed a decrease of approximately 90–99% in the specific activities and k cat values. From the plot of pH against log( k cat / K m ), the p K a values for glutathione (GSH) in the wild-type enzyme-GSH complex and the S9A-GSH complex were estimated to be 6.8 and 8.6, respectively. From these results, we suggest that the evolutionarily conserved Ser9 residue in Os GST F5 plays a major role in the catalytic mechanism of rice GST by lowering the enzyme's p K a and enhancing the nucleophilicity of the GSH thiol in the active site.

Affinity-guided covalent conjugation reactions based on PDZ-peptide and SH3-peptide interactions.

Specific protein-peptide interactions are prevalent in the living cells and form a tightly regulated signaling network. These interactions, many of which have structural information revealed, provide ideal templates for affinity-guided covalent bioconjugation. Here we report the development of a set of four new reactions that covalently and site-specifically link nonenzymatic scaffolding domains (two PDZ and two SH3 domains) and their ligands through thiol-chloroacetyl SN2 reaction. Guided by the three-dimensional structure of the wild type complex, a selected position of the protein was mutated to cysteine, and at the same time, an α-chloroacetyl group was installed at a corresponding position of the peptide. Specific binding interaction between the two brings the reactive groups into close proximity, converts the nonreactive cysteine residue into a content-dependent reactive site, and induces the nucleophilic reaction that is inert in the absence of the binding event. The specificity, orthogonality, and modularity of the four reactions were characterized, the reaction was applied to label proteins in vitro and receptor on the surface of mammalian cells, and the system was utilized to assemble covalent protein complexes with unnatural geometries.

Multiple pathways mediate luteinizing hormone regulation of cGMP signaling in the mouse ovarian follicle.

Luteinizing hormone (LH) regulation of the epidermal growth factor (EGF) network is critical for oocyte maturation and the ovulatory process. Recent studies have indicated that C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR2) play an important role in the control of meiotic arrest. Here, we investigated the involvement of the EGF network in the LH-dependent regulation of the CNP/NPR2 axis and cGMP accumulation. LH/hCG treatment causes a major decrease in both cGMP and the CNP precursor (natriuretic peptide precursor C [Nppc]) mRNA accumulation in vivo and in vitro. However, the cGMP downregulation precedes the decrease in Nppc mRNA by more than 1 h. Amphiregulin, an EGF-like factor, suppresses Nppc mRNA levels in cultured follicles to the same extent as LH, and this effect is completely prevented by the EGF receptor (EGFR) kinase inhibitor AG1478. However, the LH-dependent suppression of Nppc is insensitive to AG1478. Similarly, Nppc suppression by LH occurs in follicles from EGFR null mice. These findings document that EGFR signaling is sufficient to downregulate CNP, but is not necessary for LH action. When cGMP concentration in the follicle is measured, the short-term, but not long-term, LH effects on cGMP are prevented by AG1478, suggesting that ligand availability may be responsible for the late response. Human CG decreases the CNP-dependent cGMP synthesis in wild-type and EGFR knockdown cumulus-oocyte complexes. These findings demonstrate that redundant pathways are involved in the regulation of cGMP. EGFR-dependent events are involved in the short-term regulation of cGMP, whereas the long-term effects may involve regulation of the CNP.

New raltegravir resistance pathways induce broad cross-resistance to all currently used integrase inhibitors

The possibility of replacing raltegravir or elvitegravir with dolutegravir in heavily treatment-experienced patients failing on raltegravir/elvitegravir has been evaluated in VIKING trials. All studied patients failed by the most common pathways, Y143, Q148 and N155, and dolutegravir demonstrated efficacy except for Q148 viruses. The aim of this study was to explore, in the same way, the behaviour of dolutegravir in comparison with raltegravir and elvitegravir against the atypical resistance integrase profiles, G118R and F121Y, described in HIV-1 patients failing on raltegravir therapy. The behaviour of integrases with mutations G118R and F121Y towards raltegravir, elvitegravir and dolutegravir was analysed by evaluating phenotypic susceptibility and by means of in silico techniques (investigating binding affinities and the stabilization of the inhibitors in terms of their hydrogen bond network). The phenotypic analysis of G118R and F121Y showed high resistance to raltegravir, elvitegravir and dolutegravir with a fold change >100 when the clinically derived integrase was used, and resistance was also seen when mutations were tested alone in an NL43 backbone, but more often with a lower fold change. In silico, results showed that G118R and F121Y enzymes were associated with reduced binding affinities to each of the inhibitors and with a decreased number of hydrogen bonds compared with the wild-type complexes. This study showed that G118R and F121Y mutations, rarely described in patients failing on raltegravir, induced broad cross-resistance to all currently used integrase inhibitors. These results are in accordance with our thermodynamic and geometric analysis indicating decreased stability compared with the wild-type complexes.

Analyzing machupo virus-receptor binding by molecular dynamics simulations.

In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein–protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host–virus protein–protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein–protein interactions.

Network of Hydrogen Bonds near the Oxygen-Evolving Mn4CaO5 Cluster of Photosystem II Probed with FTIR Difference Spectroscopy

We previously provided experimental evidence that an extensive network of hydrogen bonds exists near the oxygen-evolving Mn4CaO5 cluster in photosystem II and that elements of this network form part of a dominant proton-egress pathway leading from the Mn4CaO5 cluster to the thylakoid lumen. The evidence was based on (i) the elimination of the same ν(C═O) mode of a protonated carboxylate group in the S2-minus-S1 FTIR difference spectrum of wild-type PSII core complexes from the cyanobacterium Synechocystis sp. PCC 6803 by the mutations D1-E65A, D2-E312A, and D1-E329Q and (ii) the substantial decrease in the efficiency of the S3 to S0 transition caused by the mutations D1-D61A, D1-E65A, and D2-E312A. The eliminated ν(C═O) mode corresponds to an unidentified carboxylate group whose pKa value decreases in response to the increased charge that develops on the Mn4CaO5 cluster during the S1 to S2 transition. In the current study, we have extended our work to include the ν(C═O) regions of other Sn+1-minus-Sn FTIR difference spectra and to additional mutations of residues inferred to participate in networks of hydrogen bonds near the Mn4CaO5 cluster or leading from the Mn4CaO5 cluster to the thylakoid lumen. Our data suggest that a different carboxylate group has its pKa value increased during the S2 to S3 transition and that a third carboxylate group experiences a change in its environment during the S0 to S1 transition. The pKa values that shift during the S1 to S2 and S2 to S3 transitions appear to be restored during the S3 to S0 transition. The D1-R334A mutation decreases or eliminates the same ν(C═O) modes from the S2-minus-S1 and S3-minus-S2 spectra as mutations D1-E65A, D2-E312A, and D1-E329Q and substantially decreases the efficiency of the S3 to S0 transition. We conclude that D1-R334 participates in the same dominant proton-egress pathway that was identified in our previous study. The D1-Q165E mutation leaves the ν(C═O) region of the S2-minus-S1 FTIR difference spectrum intact, but it eliminates a mode from this region of the S3-minus-S2 spectrum. We conclude that D1-Q165 participates in an extensive network of hydrogen bonds that that extends across the Mn4CaO5 cluster to the D1-E65/D2-E312 dyad and that includes D1-E329 and several water molecules including the W2 and W3 water ligands of the Mn4CaO5 cluster's dangling MnA4 and Ca ions, respectively. The D2-E307Q, D2-D308N, D2-E310Q, and D2-E323Q mutations alter the ν(C═O) regions of none of the FTIR difference spectra. We conclude that these four residues are located far from the three unidentified carboxylate groups that give rise to the ν(C═O) features observed in the FTIR difference spectra.

Investigation on the mechanism for the binding and drug resistance of wild type and mutations of G86 residue in HIV-1 protease complexed with Darunavir by molecular dynamic simulation and free energy calculation

Residue Gly86 is considered as the highly conversed residue in the HIV-1 protease. In our work, the detailed binding free energies for the wild-type (WT) and mutated proteases binding to the TMC-114 are estimated to investigate the protein-inhibitor binding and drug resistance mechanism by molecule dynamic simulations and molecular mechanics Poisson Boltzmann surface area (MM-PBSA) method. The binding affinities between the mutants and inhibitor are different than that in the wild-type complex and the major resistance to Darunavir (DRV) of G86A and G86S originate from the electrostatic energy and entropy, respectively. Furthermore, free energy decomposition analysis for the WT and mutated complexes on the basis of per-residue indicates that the mutagenesis influences the energy contribution of the residue located at three regions: active site region (residue 24-32), the flap region, and the region around the mutated residue G86 (residue 79-88), especially the flap region. Finally, further hydrogen bonds and structure analysis are carried out to detect the relationship between the energy and conformation. In all, the G86 mutations change the flap region's conformation. The experimental results are in good agreement with available results.

The Structural Basis of PI3K Cancer Mutations: From Mechanism to Therapy

While genetic alteration in the p85α-p110α (PI3K) complex represents one of the most frequent driver mutations in cancer, the wild-type complex is also required for driving cancer progression through mutations in related pathways. Understanding the mechanistic basis of the function of the phosphoinositide 3-kinase (PI3K) is essential for designing optimal therapeutic targeting strategies. Recent structural data of the p85α/p110α complex unraveled key insights into the molecular mechanisms of the activation of the complex and provided plausible explanations for the well-established biochemical data on p85/p110 dimer regulation. A wealth of biochemical and biologic information supported by recent genetic findings provides a strong basis for additional p110-independent function of p85α in the regulation of cell survival. In this article, we review the structural, biochemical, and biologic mechanisms through which p85α regulates the cancer cell life cycle with an emphasis on the recently discovered genetic alterations in cancer. As cancer progression is dependent on multiple biologic processes, targeting key drivers such as the PI3K may be required for efficacious therapy of heterogeneous tumors typically present in patients with late-stage disease.

Dynamic Effects of Tropomyosin D230N Mutation and Fetal Troponin T on the Tropomyosin Overlap Region

Introduction: Mutations in the cardiac thin filament cause changes in protein structure and dynamics. These alterations result in the complex tissue remodeling seen in patients with hypertrophic and dilated cardiomyopathies. Our group is investigating the effects of these mutations on the tropomyosin (Tm) head-to-tail overlap domain. In particular, we are investigating the alpha-Tm D230N mutation that causes a unique bimodal distribution of remodeling. We hypothesize that this is caused by the modulatory effects of the fetal form of cTnT (cTnT1) on overlap structure and function.Methods: Tm was modified to have one cysteine at residue 271 for both wildtype and D230N mutations, and was labeled with the FRET acceptor DDPM. cTnT1 or adult cardiac troponin T (cTnT3) were modified to have one cysteine at residue 100 and labeled with the FRET donor AEDANS. Labeled cTnT1 or cTnT3 were reconstituted into the troponin complex and combined with labeled Tm of either wildtype or D230N. Steady-state and lifetime data was collected.Results: The distance between the labeled sites in the wildtype complex increased with calcium activation. In contrast, the calcium activated state of the D230N complex resulted in a shorter measured distance. cTnT1 also resulted in a closer distance. cTnT1 in addition to D230N mutation resulted in a closer orientation compared D230N or cTnT1 alone. Differences in measured distances caused by the variants were more pronounced in the calcium activated state than in the low calcium state.Conclusion: The increased distance measured with wildtype complex supported the increased flexibility of Tm accompanying thin filament activation. The decreased distance measured at the Tm overlap supports the hypothesis that cTnT1 interacts with D230N in a manner that amplifies the effects of the mutation alone at the Tm overlap region.

Crystal Structure of Synechocystis Mutants and Plant Photosystem I

Plant Photosystem I (PSI) is one of the most intricate membrane complexes in Nature. It comprises two loosely bound reaction center and light-harvesting (LHC) complexes containing 18 subunits 174 chlorophylls, 32 carotenoids 2 quinines and 3 iron-sulfur clusters. The crystal structure at 3.1 A resolution reported her add several new features that were not detected in the previous structures. The location of an additional ten β-carotenes as well as five chlorophylls and several loop regions are now modeled. This represents the most complete plant Photosystem I structure obtained thus far, revealing the locations of and interactions among 18 protein subunits and 211 non-covalently bound photochemical cofactors. We also detected 12 additional carotenoids especially in the LHC complex as well as structural lipids. The latter suggest evolutionary scenario where Lhca1 and Lhca2 coevolved with LHCII and Lhca4 coevolved with CP29.Recently an operon encoding PSI was identified in cyanobacterial marine viruses. A PSI that mimics the most important feature was generated in Synechocystis 6803. This PSI is promiscuous for its electron donor and can accept electrons from respiratory cytochromes. The wild type and promiscuous PSI complexes were isolated, crystallized and their structure was solved to 4 and 3.8 A resolution, respectively. In addition we solved the virus-like structure of PsaL and PsaI minus PSI mutant at 2.8 A resolution. The novel structure of PSI from mesophilic cyanobacterium is different in several aspects in comparison with the previously published structure of PSI from Thermosynechococcus elongatus. The common properties of mesophilic cyanobacterial and plant PSI will be discussed.

The PS1 Hairpin of Mcm3 Is Essential for Viability and for DNA Unwinding In Vitro

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 K499A strain. The ATPase activity of Mcm2-73K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.