Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.     

Rhythmic Conformational Change in a SinglePolymer Chain Induced by Laser Irradiation

We have investigated a rhythmic conformational change in a single polymer chain (T4DNA, 166 kbp, the contour length = 56 m) between a folded state and a elongated state under thermodynamically open conditions using a focused continuons wave (cw) Nd:YAG laser beam (wavelength = 1064 nm), where the focused laser beam plays dual roles both of trapping of a polymer chain at the focus and making temperature gradient there. The oscillatory phenomenon is discuseed in terms of a limit-cycle oscillation with dissipation of photon energy.     

Covalent Binding and Conformational Change of pUC19 DNA by Rhodium (II) Metal Complex

Abstract

Binding of Rhodium (II) acetate [Rh₂(O₂CCH₃)₄] (Rh1) compound with plasmid pUC19 DNA has been studied using different molar ratio of Rh1. After incubation for 24hr at 37 °C, binding of the Rh1 to pUC19 DNA was confirmed by agarose gel electrophoresis. The electrophoretic results indicated the slower migration speed for the linearized pUC19 DNA. Conformation change of the DNA after Rh1 binding was also indicated at higher molar ratio of Rh1. The atomic force microscopy images showed that the Rh1 induced the conformation change to unwind pUC19 DNA. The Rh1-DNA complexes are observed very stable due to covalent bond. This study clearly demonstrates that [Rh₂(O₂CCH₃)₄] reacts with pUC19 DNA and covalently binds to be stable Rh1-pUC19 DNA as interstrand adducts.     

Conformational State-Dependent Anion Binding in Prestin: Evidence for Allosteric Modulation

Outer hair cells boost auditory performance in mammals. This amplification relies on an expansive array of intramembranous molecular motors, identified as prestin, that drive somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, we are able to assess prestin's conformational state and interrogate the effectiveness of anions on prestin's activity. We find that the affinity of anions depends on the state of prestin that we set with a variety of perturbations (in membrane tension, temperature, and voltage), and that movement into the expanded state reduces the affinity of prestin for anions. These data signify that anions work allosterically on prestin. Consequently, anions are released from prestin's binding site during expansion, i.e., during hyperpolarization. This is at odds with the extrinsic voltage sensor model, which suggests that prestin-bound intracellular anions are propelled deep into the membrane. Furthermore, we hypothesize that prestin's susceptibility to many biophysical forces, and notably its piezoelectric nature, may reflect anion interactions with the motor.     

From Induced Fit to Conformational Selection: A Continuum of Binding Mechanism Controlled by the Timescale of Conformational Transitions

In receptor-ligand binding, a question that generated considerable interest is whether the mechanism is induced fit or conformational selection. This question is addressed here by a solvable model, in which a receptor undergoes transitions between active and inactive forms. The inactive form is favored while unbound but the active form is favored while a ligand is loosely bound. As the active-inactive transition rates increase, the binding mechanism gradually shifts from conformational selection to induced fit. The timescale of conformational transitions thus plays a crucial role in controlling binding mechanisms.     

Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Binding of Lipoic Acid Induces Conformational Change and Appearance of a New Binding Site in Methylglyoxal Modified Serum Albumin

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.     

Evidence for a Conformational Change in Subunit III of Bovine Heart Mitochondrial Cytochrome c Oxidase1

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl—biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.     

Genetic Evidence for Functional Interactions between Actin Noncomplementing (Anc) Gene Products and Actin Cytoskeletal Proteins in Saccharomyces Cerevisiae

We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.     

Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.     

Amer1/WTX与APC相互作用的新结合位点鉴定

Amer1 (APC membrane recruitment protein 1)又称为WTX（Wilms’ tumor X）,是首个发现位于X染色体上的抑癌基因．由于其定位的特殊性,Amer1近年来成为研究的热点之一．研究表明,Amer1作为骨架蛋白在细胞内与多种蛋白（APC,β-catenin,Axin等）直接结合,在Wnt信号通路中发挥着重要功能．据报道Amer1/WTX含有3个肿瘤抑制蛋白质APC (adenomatous polyposis coli, APC)的结合位点,对于APC在细胞膜上的定位过程中发挥着重要的作用．但是,通过序列比对发现,Amer1可能存在第4个被忽略的APC的结合位点,定位于A1和A2之间．为了验证该片段能否与APC结合,分别构建了GST-Amer1 (365-375)和His-APC (407+775)两种重组蛋白．通过GST-pull down,证明了这两个片段存在相互作用,并进一步通过ITC (isothermal titration calorimetry) 实验测定了两者结合的亲和力．本研究结果不仅在体外证实了Amer1第4个APC结合位点的存在,也为APC和 Amer1/WTX复合物的结构和功能的研究打下了良好的基础．     

Conformational Changes Induced in the Human Protein Translin and in the Single-stranded Oligodeoxynucleotides d(GT)12 and d(TTAGGG)5 Upon Binding of These Oligodeoxynucleotides by Translin

Abstract

Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)_n, the human telomeric repeats, d(TTAGGG)_n, and the Tetrahymena telomeric repeats, d(GGGGTT)_n. These data suggested that translin might be involved in recombination at d(GT)_n·d(AC)_n microsatellites and in telomere metabolism [E. Jacob, L. Pucshansky, E. Zeruya, N. Baran, H. Manor. J. Mol. Biol. 344, 939–950 (2004), S. Cohen, E. Jacob, H. Manor. Biochim. Biophys. Acta. 1679, 129–140 (2004)]. Other data indicated that translin might stimulate binding of telomerase to single- stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats [S. Cohen, E. Jacob, H. Manor. Biochim. Biophys. Acta. 1679, 129–140 (2004)]. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT) and d(TTAGGG)5. We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)12, the fraction of a-helices decreases from ～67% to ～50%, while the fraction of turns and of the unordered structure increases from ～11% to ～17% and from ～19% to ～24%, respectively. In the bound oligodeoxynucleotide d(GT), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)5 formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.     

Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.     

Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166''s participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.     

A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface

Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing.     

Actin detected in Mouse Neuroblastoma Cells by Binding of Heavy Meromyosin

HEAVY meromyosin (HMM) fragments of myosin from striated muscle specifically bind with actin filaments to form complexes that are readily observed by electron microscopy¹ in both negatively-stained preparations and sectioned material. The composite or “decorated filaments” appear like a line of arrowheads. The existence of such decorated filaments in cells or some cell fraction after treatment with HMM indicates that actin is present. Ishikawa et al.² used this to demonstrate actin in a number of cultured cell types. More recently, other workers have similarly demonstrated actin filaments in slime mould³, amoebae^4,5, blood platelets⁶, microvilli⁷, macrophages⁸ and, less convincingly, in sperm tails⁹ and the mitotic spindle¹⁰. We prove here that filaments from the cortical region of mouse neuroblastoma cells bind HMM and therefore contain actin.     

Conformational Change in MSH2-MSH6 upon Binding DNA Coupled to ATPase Activity

Postreplication DNA mismatch repair is initiated by the eukaryotic protein MSH2-MSH6 or the prokaryotic protein MutS, both showing overall conserved structure and functionality. Crystal structures of MSH2-MSH6 and MutS bound to the mismatch DNA reveal a closed architecture of the clamp and the lever domains exhibiting strong contacts with the bent DNA backbone. Long molecular dynamics simulations of the human MSH2-MSH6 protein in the absence of a DNA show an altered conformation of the protein that reflects the protein's state before binding to DNA. The clamp and the lever domains of both MSH6 and MSH2 open in an asymmetric and dramatic fashion. The opening of the clamp and the lever domains in the absence of DNA is coupled to changes in the ATPase domains, which explains the experimentally observed diminished ATPase activity in DNA-free MSH2-MSH6 and illustrates the allosteric coupling between DNA binding and ATPase activity.     

Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved.     

Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.