Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.     

DNA Dynamics during Early Double-Strand Break Processing Revealed by Non-Intrusive Imaging of Living Cells

Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks (DSB) is homologous recombination (HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and -targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.     

Spatio-Temporal Dynamics of Chromatin Containing DNA Breaks

The cellular response to DNA breaks consists of a complex signaling network that coordinates the initial recognition of the lesion with the induction of cell cycle checkpoints and DNA repair. With DNA wrapped around histone proteins and packaged into higher order levels of chromatin structure, the detection of a single DNA break (DSB) in the genome is the molecular equivalent of finding a needle in a haystack . A recent study from our laboratory used high-resolution electron microscropy and live cell imaging to demonstrate that chromatin undergoes a marked reorganization in response to a DSB. In an energy dependent manner, chromatin rapidly decondenses to a more open configuration in the regions surrounding the lesion. We propose that this ATP dependent chromatin remodeling event facilitates the subsequent recognition and processing of damaged DNA. While the chromatin surrounding the lesion remodels to a more open configuration, the DNA break itself remains relatively immobile over time, consistent with the idea that DNA damage response proteins migrate to positionally stable sites of damaged DNA 1. The lack of significant movement of chromatin regions containing DSBs has implications for the process by which chromosomal translocations form.     

The Gating Mechanism of the Human Aquaporin 5 Revealed by Molecular Dynamics Simulations

Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.     

A Model of Lipid-Free Apolipoprotein A-I Revealed by Iterative Molecular Dynamics Simulation

Apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, has been proven inversely correlated to cardiovascular risk in past decades. The lipid-free state of apo A-I is the initial stage which binds to lipids forming high-density lipoprotein. Molecular models of lipid-free apo A-I have been reported by methods like X-ray crystallography and chemical cross-linking/mass spectrometry (CCL/MS). Through structural analysis we found that those current models had limited consistency with other experimental results, such as those from hydrogen exchange with mass spectrometry. Through molecular dynamics simulations, we also found those models could not reach a stable equilibrium state. Therefore, by integrating various experimental results, we proposed a new structural model for lipid-free apo A-I, which contains a bundled four-helix N-terminal domain (1–192) that forms a variable hydrophobic groove and a mobile short hairpin C-terminal domain (193–243). This model exhibits an equilibrium state through molecular dynamics simulation and is consistent with most of the experimental results known from CCL/MS on lysine pairs, fluorescence resonance energy transfer and hydrogen exchange. This solution-state lipid-free apo A-I model may elucidate the possible conformational transitions of apo A-I binding with lipids in high-density lipoprotein formation.     

A Possible Binding Path of Ergosterol Within Elicitins Revealed by Molecular Dynamics

Abstract

Elicitins, produced by most of the phytopathogenic fungi of the genus Phytophthora, provoke in the tobacco plant both remote leaf necrosis and the induction of a resistance against subsequent attack by various micro-organisms. The crystal structure of b-cryptogein (CRY), secreted by Phytophthora cryptogea, was previously reported as well as the first structure of a SCP/sterol complex, the ergosterol-complexed, mutated CRY (K13H). In K13H, the ergosterol molecule is encapsulated in a large internal hydrophobic cavity which is not present in CRY. This binding induces a minor conformational change in the protein structure. Molecular dynamics studies were undertaken to precise the structural behaviour of CRY and K13H with respect to the complexation of the ergosterol.

Although it is not possible to simulate the entrance of the ergosterol in the protein, we assume that capture and release of the ligand possibly both occur following the same path. Our results show that, in the complex K13H, the ergosterol molecule is pushed towards the residue 13 which play a key role in the necrotic activity of the protein. It is likely that the polarity of residue 13, favouring the binding of the hydroxy l of the ligand, would be involved in the recognition of the sterol and in an optimisation of its orientation. Thus, in a first step, the molecule of ergosterol would be rotated around itself to a position which makes possible, in a second step, its translation to the internal cavity, as a key in a keyhole.     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

Chromatin Accessibility Dynamics during Chemical Induction of Pluripotency

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Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln⁴¹ and Leu⁸¹ as DNA lesion sensors.     

海桑属植物分子鉴定的ISSR分析

为准确鉴别海桑属植物,采用ISSR（inter-simple sequence repeat）分子标记技术,对6种海桑属植物(海桑、拟海桑、杯萼海桑、海南海桑、卵叶海桑及无瓣海桑)基因组DNA进行PCR扩增。建立了海桑属植物ISSR标记的标准程序和海桑属各物种的DNA指纹数据库。采用的11个引物共产生71个物种特征性标记,其中无瓣海桑23个,海南海桑16个,海桑15个,杯萼海桑6个,拟海桑6个,卵叶海桑5个。运用这些特征性标记,可迅速区分海桑属植物。研究表明ISSR技术是在分子水平上鉴定海桑属植物的一种行之有效的方法。     

Chromatin Regulation of DNA Damage Repair and Genome Integrity in the Central Nervous System

With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.