Heterodimeric Structure Research Articles

Structural Basis for Conserved Regulation and Adaptation of the Signal Recognition Particle Targeting Complex

The signal recognition particle (SRP) is a ribonucleoprotein complex with a key role in targeting and insertion of membrane proteins. The two SRP GTPases, SRP54 (Ffh in bacteria) and FtsY (SRα in eukaryotes), form the core of the targeting complex (TC) regulating the SRP cycle. The architecture of the TC and its stimulation by RNA has been described for the bacterial SRP system while this information is lacking for other domains of life. Here, we present the crystal structures of the GTPase heterodimers of archaeal (Sulfolobus solfataricus), eukaryotic (Homo sapiens), and chloroplast (Arabidopsis thaliana) SRP systems. The comprehensive structural comparison combined with Brownian dynamics simulations of TC formation allows for the description of the general blueprint and of specific adaptations of the quasi-symmetric heterodimer. Our work defines conserved external nucleotide-binding sites for SRP GTPase activation by RNA. Structural analyses of the GDP-bound, post-hydrolysis states reveal a conserved, magnesium-sensitive switch within the I-box. Overall, we provide a general model for SRP cycle regulation by RNA.

Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications.

The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring.

Inhibition of HIV-1 Reverse Transcriptase Dimerization by Small Molecules.

Because HIV-1 reverse transcriptase is an enzyme whose catalytic activity depends on its heterodimeric structure, this system could be a target for inhibitors that perturb the interactions between the protein subunits, p51 and p66. We previously demonstrated that the small molecule MAS0 reduced the association of the two RT subunits and simultaneously inhibited both the polymerase and ribonuclease H activities. In this study, some analogues of MAS0 were rationally selected by docking studies and evaluated in vitro for their ability to disrupt dimeric assembly. Two inhibitors were identified with improved activity compared to MAS0. This study lays the basis for the rational design of more potent inhibitors of RT dimerization.

Computationally Designed Bispecific Antibodies using Negative State Repertoires

A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form. This is a difficult problem because mutations predicted to destabilize the negative state might be accommodated by small conformational rearrangements. To overcome this challenge, we employ an iterative strategy that cycles between sequence design and protein docking in order to build up an ensemble of alternative negative state conformations for use in specificity prediction. We have applied our technique to the design of heterodimeric CH3 interfaces in the Fc region of antibodies. Combining computationally and rationally designed mutations produced unique designs with heterodimer purities greater than 90%. Asymmetric Fc crystallization was able to resolve the interface mutations; the heterodimer structures confirmed that the interfaces formed as designed. With these CH3 mutations, and those made at the heavy-/light-chain interface, we demonstrate one-step synthesis of four fully IgG-bispecific antibodies.

Facing the challenge of a functional characterization of toll-like receptor (TLR)1 and TLR2 in common carp

Toll-like receptors are a family of germline-encoded pattern recognition receptors which activate rapid inflammatory responses upon detection of their cognate ligands. In mammals, TLR2 can form a heterodimer with TLR1, 6, or 10, and recognize lipoproteins/lipopeptides from Gram-positive bacteria. Of these TLRs, only tlr1 and tlr2 are present in fish genomes. In the carp genome, a single full-length tlr1 gene could be identified, whereas two functional genes for carp tlr2 exist. High expression of tlr1 and both tlr2 genes was found in immune organs and leukocytes of both myeloid and lymphoid origin. Of the two tlr2 genes, tlr2a was always higher expressed than tlr2b. Three-dimensional modelling of the carp Tlr1 and Tlr2 proteins appear to confirm the ability to form a heterodimer, with a pocket that can accommodate the tri-acylated lipopeptide ligand Pam3CSK4, similar to the heterodimer structure of human TLR1-TLR2 on which the model was built. In cell lines of human as well as fish origin transfected to overexpress the carp Tlr proteins, co-localization of carp Tlr1 and Tlr2 could be revealed with confocal microscopy. We were unable to confirm Tlr localization to the cell surface, the expected sub-cellular localization of Tlr1 and Tlr2. Further experimental evidence would be needed to unequivocally prove molecular interaction between the two Tlr proteins. In previous work, we have described ligand-specific activation of carp Tlr2 overexpressed in human cells (HEK), via measurement of increased phosphorylation levels of the MAP kinase p38 by Western blot. Instead of this semi-quantitative method, here we used a read-out system based on NF-κB activation and subsequent measurement of luminescence. This quantitative method could not confirm our initial ligand binding studies, not using human cell lines (HEK, HeLa), nor using a fish cell line (EPC). We will discuss possible explanations for the unsuccessful ligand-specific activation of NF-κB after overexpression of Tlr1 and/or Tlr2 in human, but also fish cell lines, to propose alternative future strategies for studying ligand binding properties of fish Tlrs.

Selective formation of Ag domains on MnO nanooctapods for potential dual imaging probes

In the present study, the growth of Ag domains on the surface of MnO octapods yielded a uniform MnO octapod–Ag heterodimer structure by a phase-transfer protocol in toluene. The Ag domains were selectively formed on one of the high index surfaces of the MnO octapods. The resulting MnO–Ag heterodimers have basically two independent domains. On this basis, multiple functions are expected, including plasmon light scattering from the Ag domains and magnetic resonance from the MnO domains. Notably, the heterodimers strongly scattered visible light at 420 nm, and even a single particle signal could be detected in a dark-field spectroscopy image. The scattering peak was extended to the near-IR range by the formation of MnO octapod–AgAu hollow heterodimers via a galvanic replacement reaction. The heterodimers also showed weak ferromagnetism at low temperature, and exhibited a positive T1 signal in magnetic resonance imaging. These properties demonstrate that the MnO–Ag heterodimers can potentially serve as dual imaging probes for biological systems.

Analysis of Castor by ELISAs that Distinguish Ricin and Ricinus communis agglutinin (RCA)

AbstractTo facilitate the analysis of castor (Ricinus communis L.) seed fractions and germplasm for ricin content, we investigated the use of enzyme‐linked immunosorbent assay (ELISA) methods to differentiate between ricin toxin and the related Ricinus communis agglutinin (RCA). Both proteins are based on a heterodimeric AB structure, with a common A chain. RCA comprises two dimers of A and B chains. Both proteins are found in the meal remaining after castor oil extraction and impede the commercial production of castor seed in the USA. We identified pairs of monoclonal antibodies (mAbs) that could distinguish between the structurally related proteins that share a common A chain. Antibody specificity was determined by ELISA and checked by immunoblotting. We found that mAb–mAb pairs afforded quantification of each castor protein, and that a mAb paired with a commercial polyclonal antibody provided detection of both with comparable sensitivity.

Concerted removal of the Erb1-Ytm1 complex in ribosome biogenesis relies on an elaborate interface.

The complicated process of eukaryotic ribosome biogenesis involves about 200 assembly factors that transiently associate with the nascent pre-ribosome in a spatiotemporally ordered way. During the early steps of 60S subunit formation, several proteins, collectively called A3 cluster factors, participate in the removal of the internal transcribed spacer 1 (ITS1) from 27SA3 pre-rRNA. Among these factors is the conserved hetero-trimeric Nop7–Erb1–Ytm1 complex (or human Pes1–Bop1–Wdr12), which is removed from the evolving pre-60S particle by the AAA ATPase Rea1 to allow progression in the pathway. Here, we clarify how Ytm1 and Erb1 interact, which has implications for the release mechanism of both factors from the pre-ribosome. Biochemical studies show that Ytm1 and Erb1 bind each other via their ß-propeller domains. The crystal structure of the Erb1–Ytm1 heterodimer determined at 2.67Å resolution reveals an extended interaction surface between the propellers in a rarely observed binding mode. Structure-based mutations in the interface that impair the Erb1–Ytm1 interaction do not support growth, with specific defects in 60S subunit synthesis. Under these mutant conditions, it becomes clear that an intact Erb1–Ytm1 complex is required for 60S maturation and that loss of this stable interaction prevents ribosome production.

Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition

Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1-175 and 50-292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59-87) embraces pUL50 and contributes 1510 Å(2) to the total interface area (1880 Å(2)). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.

The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins.

GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin-Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.

Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses.

Higher-Order Assembly of BRCC36–KIAA0157 Is Required for DUB Activity and Biological Function

SummaryBRCC36 is a Zn2+ dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN+ domain protein BRCC36 associates with pseudo-DUB MPN− proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide the first explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner, and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function.

Evolutionary and structural analyses of heterodimeric proteins composed of subunits with same fold.

Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non-redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23-24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co-localized in the cell.

Differences in solution dynamics between lens β-crystallin homodimers and heterodimers probed by hydrogen–deuterium exchange and deamidation

BackgroundLens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. βB2 crystallin in the lens of the eye readily forms dimers with other β-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. MethodsStructures of βA3 and βB2 crystallin homodimers and the βA3/βB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen–deuterium exchange with mass spectrometry. We further mimicked deamidation in βB2 and probed the effect on the βA3/βB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. ResultsBoth βA3 and βB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of βB2 was supported by the identification of chemical crosslinks between lysines in βB2 within the heterodimer that were inconsistent with βB2's extended homodimeric structure. The compact structure of βA3 was supported by an overall decrease in mobility of βA3 in the heterodimer detected by NMR.In βB2, peptides 70–84 and 121–134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in βA3, 97–109 and 134–149, had 25–50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between βB2 and βA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in βB2 at this predicted interface led to a less compact βB2 in the heterodimer.The more compact structure of the βA3/βB2 heterodimer was also more heat stable than either of the homodimers. ConclusionsThe major structural proteins in the lens, the β-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of βB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. General significanceUnderstanding complex hetero-oligomer interactions between β-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis.

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE-PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25-PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.

Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly.

Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. KM values were 1.59 and 0.53 mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. Vmax values for CDNB and GSH were also determined as 5.58 and 1.88 EU/mL, respectively. In addition, inhibition effects of Ag+, Cu2+, Cd2+, Fe3+, Pb2+, Cr2+, Co2+ and Zn2+ metal ions were investigated on the enzyme activity and IC50, Ki values were calculated for these metal ions.

Characterization of a Putative Fanconi Anemia D2 Protein Histone‐Binding Domain and Chromodomain

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure and heightened cancer susceptibility. FA is caused by biallelic mutations in 16 genes. The FA proteins function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of the FA-BRCA pathway is the site-specific monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to chromatin, where they recruit several structure-specific endonucleases. However, how these proteins are targeted to chromatin remains undetermined. We recently uncovered sequence homology between FANCD2 and the histone H4 binding domain (HBD) of D. melanogaster p55, a component of the CAF1 and NuRD chromatin remodeling complexes. In silico analysis of this region uncovered a putative methyl-lysine-binding chromodomain (CD), with sequence homology to the chromodomains of HP1a and TIP60. In a histone peptide array screen, a FANCD2 fragment harboring the HBD and CD bound to unmodified, mono-, di-, and tri-methylated H4K20. These findings were verified using in vitro peptide pull-down assays. Guided by sequence conservation and molecular modeling of the crystal structure of the mouse Fancd2-Fanci heterodimer, we engineered FANCD2 lentiviral vectors harboring missense mutations in several putative key HBD/CD residues, and stably transduced FA-D2 (FANCD2-/-) patient-derived cells. As assessed by cytotoxicity assays, chromosome analysis, and G2-M-phase accumulation, mutation of the FANCD2 HBD/CD impairs FANCD2 function in ICL repair. Our studies describe an important chromatin reader domain in a FA protein and link this important human genetic disease to the regulation of chromatin plasticity.

Microtubules dual chemo and thermo-responsive depolymerization.

The effects of chemotherapeutic agent vinblastine versus low temperature of 277 K were investigated on the structure of αβ-tubulin heterodimer by means of molecular dynamics simulations. Individual experiments have shown that the vinblastine-bound heterodimer, and its apo structure under low temperature of 277 K, both undergo conformational changes toward destabilization of the dimer as compared to the apo tubulin at 300 K. Both factors exhibited weakening of the longitudinal interactions of tubulin heterodimer through displacing dimer interfacial segments, resulting in dominant electrostatic repulsion at the interface of the subunits. The two independent factors of temperature and anti-mitotic agent facilitate alteration of secondary structure in functional segments such as H1-S2 loop, H3, H10 helices, and T7 loop, which are known to be important in either longitudinal or lateral contacts among αβ-heterodimers in MTs protofilaments and their depolymerization mechanism.