12 research outputs found
Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis
Background: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process
An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering
Abstract: Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small- molecule fragment soaks. New modeling approaches reveal ’hidden’ low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function
Studies in bacterial phosphotriesterase evolution, dynamics, and engineering
the author deposited 12/06/201
Framework pro získání a analýzu apo- a holo- form proteinů z PDB
We developed a software framework that allows the analysis of ligand-free (apo) and ligand-bound (holo) forms of proteins that are accessible in PDB. The software downloads the current version of the PDB, divides the structures into groups of the same molecules, and these into apo and holo forms. Finally, it is possible to analyze pairs of apo and holo structures with respect to their different structural characteristics. In addition to the software work itself, we also verify results against previous work on an equivalent dataset, and obtain results for the current version of PDB. Keywords: protein; structural bioinformatics; PDBVyvinuli jsme softwarový framework, který umožňuje analyzovat a porovnávat apo (bez ligandu) a holo (s ligandem) strukturní formy proteinů přístupných v PDB. Software stáhne aktuální verzi PDB, rozdělí struktury do skupin stejných molekul a rozliší zda se jedná o apo či holo strukturní formu. Nakonec je možné analyzovat dvojice apo a holo struktur s ohledem na jejich odlišné strukturální charakteristiky. Kromě samotné softwarové práce prezentujeme výsledky na datasetu z výchozí výzkumu a ověřujeme je. Získáváme výsledky na aktuální verzi PDB. Klíčová slova: protein; strukturní bioinformatika; PDBDepartment of Cell BiologyKatedra buněčné biologiePřírodovědecká fakultaFaculty of Scienc
Structural Mechanisms of the Sliding Clamp and Sliding Clamp Loader: Insights into Disease and Function: A Dissertation
Chromosomal replication is an essential process in all life. This dissertation highlights regulatory roles for two critical protein complexes at the heart of the replication fork: 1) the sliding clamp, the major polymerase processivity factor, and 2) the sliding clamp loader, a spiral-shaped AAA+ ATPase, which loads the clamp onto DNA.
The clamp is a promiscuous binding protein that interacts with at least 100 binding partners to orchestrate many processes on DNA, but spatiotemporal regulation of these binding interactions is unknown. Remarkably, a recent disease-causing mutant of the sliding clamp showed specific defects in DNA repair pathways. We aimed to use this mutant as a tool to understand the binding specificity of clamp interactions, and investigate the disease further. We solved three structures of the mutant, and biochemically showed perturbation of partnerbinding for some, but not all, ligands. Using a fission yeast model, we showed that mutant cells are sensitive to select DNA damaging agents. These data revealed significant flexibility within the binding site, which likely regulates partner binding.
Before the clamp can act on DNA, the sliding clamp loader places the clamp onto DNA at primer/template (p/t) junctions. The clamp loader reaction couples p/t binding and subsequent ATP hydrolysis to clamp closure. Here we show that composition (RNA vs. DNA) of the primer strand affects clamp loader binding, and that the order of ATP hydrolysis around the spiral is likely sequential. These studies highlight additional details into the clamp loader mechanism, which further elucidate general mechanisms of AAA+ machinery
Structure, function, evolution and inhibition studies of the organophosphate detoxifying enzyme αE7
Insecticide resistance is a global concern that threatens human health and agricultural productivity. Understanding the molecular basis of resistance will help to manage future insecticide use to ensure that effective, safe and inexpensive pest control is available. In the Australian sheep blowfly Lucilia cuprina, a single mutation (Gly137Asp) in the αE7 carboxylesterase gives rise to resistance by converting the enzyme into an organophosphate (OP) hydrolase. This emergence of new activity provides a unique opportunity to investigate the molecular basis for enzyme evolution. In this thesis, I investigated the structure, function, evolution and inhibition of αE7. Chapter two describes the role of structural diversity in the function of wild type αE7. I applied new methods for extracting information about structural diversity from X-ray diffraction data to explore the changes in structure that accompany high affinity OP binding in αE7. In chapter three, I investigated the molecular basis for the evolution of catalytic OP detoxification in the blowfly. I determined the structure of the Gly137Asp variant by X-ray crystallography, which, along with molecular dynamics simulations and enzyme activity assays, revealed the role of Asp137 in the new catalytic mechanism. The new sidechain is disordered, and potentially only displays a fraction of its catalytic potential. Chapter four explores this catalytic potential through the laboratory-directed evolution of αE7 for increased OP hydrolase activity. I performed detailed kinetic and structural analysis of the evolutionary trajectory and characterized the structural changes responsible for the 8000-fold increase in OP hydrolase activity. The analysis unmasked a hidden, catalytically relevant, conformation of the active site. Furthermore, the results revealed the role of conformational diversity in the evolutionary optimization of αE7 and highlight the challenges to satisfying the competing demands of substrate binding and catalysis in
the tightly packed environment of an enzyme’s active site. This work establishes that only a fraction of the evolutionary potential of αE7 has been explored in nature. In chapter five, I combined structural knowledge of αE7 with a computational screen to discover new potent and selective inhibitors of αE7. These compounds, based on a boronic acid scaffold, act as synergists to reduce the amount of OP required to kill L. cuprina by up to 16-fold, and abolish resistance. The broad-spectrum potential for
the compounds as a new class of synergist was demonstrated by their low toxicity to animals and their ability to potentiate OP insecticides against another common insect pest, the peach-potato aphid Myzus persicae. These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy. More broadly, this thesis makes contributions to characterizing structural protein heterogeneity using X-ray diffraction, to understanding the molecular basis of enzyme evolution and to the use of in silico screens for the discovery of enzyme
inhibitors. The results from this thesis will assist the of control insect pests and the management of insecticide resistance
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Investigation of Ribonuclease HI handle region dynamics using Solution-state nuclear magnetic resonance spectroscopy, Molecular Dynamic simulations and X-ray crystallography
Ribonuclease HI (RNase HI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. The enzyme has roles in replication, genome maintenance, and is the C-terminal domain of retroviral multi-domain reverse transcriptase (RT) proteins. Murine Leukemia Virus (MLV) and Human Immunodeficiency Virus (HIV) are two such retroviruses and their RNase HI (RNHI) domains are necessary for viral replication, making it an attractive drug target. RNase HI has a “handle region”, an extended loop with a large cluster of positive residues, that is critical for substrate recognition. MLV-RNHI is active in isolation and contains a handle region, but, HIV-RNHI is inactive in isolation and does not contain a handle region. HIV-RT, however, has a region in its polymerase domain (positive charge cluster and aromatic cluster) that makes contact with the RNHI domain that may be serving as a “pseudo” handle region; additionally, insertion of a handle region into isolated HIVRNHI restores its activity. Overall, a breadth of information exists on this region’s dynamics, but important gaps remain unfilled; gaps that may potentially lead to creating effective drugs to treat the above-mentioned viruses.
Solution-state nuclear magnetic resonance (NMR) spectroscopy combined with Molecular Dynamic (MD) simulations suggest a model in which the extended handle region domain of the mesophilic Escherichia coli RNHI (EcRNHI) populates "open" (substrate-bindingcompetent) and "closed" (substrate-binding incompetent) states, while the thermophilic Thermus thermophilus RNHI (TtRNHI) mainly populates the closed state at 300 K. In addition, an in silico designed mutant Val98Ala (V98A) EcRNHI was predicted to populate primarily the closed state. Understanding the structural features and internal motions that lead RNase HI to adopt these various conformers is of central importance to better understanding RNase HI’s role in retroviral infection.
To formulate a comprehensive model on handle region dynamics, an integrative approach of NMR spectroscopy, X-ray crystallography, and MD simulations is employed. The sensitivity to internal conformational dynamics at multiple time scales of NMR spectroscopy, molecular range and resolution of X-ray crystallography, and structural interpretations of dynamic processes by MD simulations create a synergistic trio capable of tackling this issue. First, the in silico 2-state Kinetic model is validated through NMR observables that correlate with the respective conformers, thus serving as experimental analogs. The NMR parameters also correlate with the Michaelis constants (KM) for RNHI homologs and help to confirm the in silico predictions of V98A EcRNHI. This study shows the important role of the handle region in modulation of substrate recognition. It also illustrates the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function.
Next, a deeper dive is taken into handle region dynamics, specifically focusing on residue 88 and the impact its identity has on this region. Its sidechain interactions are shown to directly correlate with handle region conformations and helps to amend the originally proposed in silico 2-state Kinetic model. Lastly, looking at RNHI handle region dynamics through an evolutionary lens opens the door to uncovering novel mutations that have been previously overlooked or not identified. Through a phylogenetic analysis, researchers have reconstructed seven ancestral RNHI mutants and three of them have been expressed here. The sequence identity of these three ancestral mutants range from 60-87% to extant homologs and this is reflected by similar peak positions in their 15N HSQC spectra. Requisite experiments to assign the NMR backbone have been completed