44 research outputs found
Polyglutamine monomer structure and its implications for molecular self-assembly
Polyglutamine is a naturally occurring peptide found within several proteins in
neuronal cells of the brain, and its aggregation has been implicated in several
neurodegenerative diseases, including Huntington's disease. The resulting aggregates
have been demonstrated to possess ~-sheet structure, and aggregation has been shown to
start with a single misfolded peptide. The current project sought to computationally
examine the structural tendencies of three mutant poly glutamine peptides that were
studied experimentally, and found to aggregate with varying efficiencies. Low-energy
structures were generated for each peptide by simulated annealing, and were analyzed
quantitatively by various geometry- and energy-based methods. According to the results,
the experimentally-observed inhibition of aggregation appears to be due to localized
conformational restraint placed on the peptide backbone by inserted prolines, which in
tum confines the peptide to native coil structure, discouraging transition towards the ~sheet
structure required for aggregation. Such knowledge could prove quite useful to the
design of future treatments for Huntington's and other related diseases
Exploring the structural basis of conformational heterogeneity and autoinhibition of human cGMP-specific protein kinase Iα through computational modelling and molecular dynamics simulations.
Protein kinase Iα (PKGIα) is a pivotal cyclic guanosine monophosphate (cGMP) signalling protein. Major steps related to the structural plasticity of PKGIα have been inferred but the structural aspects of the auto-inhibition and multidomain tertiary organization of human PKGIα in active and inactive form are not clear. Here we combine computational comparative modelling, protein-protein docking and molecular dynamics (MD) simulations to investigate structural details of the repressed state of the catalytic domain of PKGIα. Exploration of the potential inhibitory conformation of the auto-inhibitory domain (AI) within the catalytic cleft reveals that the pseudo-substrate motif binds with residues of the glycine rich loop and substrate-binding lobe. Dynamic changes as a result of coupling of the catalytic and AI domains are also investigated. The three-dimensional homodimeric models of PKGIα in the active and inactive state indicate that PKGIα in its inactive-state attains a compact globular structure where cyclic nucleotide binding (CNB-A/B) domains are buried, whereas the catalytic domains are inaccessible with their substrate-binding pockets facing the N-terminal of CNB-A. Contrary to this, the active-state model of PKGIα shows an extended conformation where CNB-A/B domains are slightly rearranged and the catalytic domains of homodimer flanking the C-terminal with their substrate binding lobes free to entrap downstream proteins. These findings are consistent with previously reported static images of the multidomain organization of PKGIα. Structural insights pertaining to the conformational heterogeneity and auto-inhibition of PKGIα provided in this study may help to understand the dynamics-driven effective regulation of PKGIα
Probing ligand selectivity in pathogens
Why does protein kinase A respond to purine nucleosides in certain pathogens, but not to the cyclic nucleotides that activate this kinase in most other organisms
Structural Basis of Tonic Inhibition by Dimers of Dimers in Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels
The hyperpolarization-activated cyclic-nucleotide-modulated
(HCN)
ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic
AMP (cAMP) modulates HCN activity through the cAMP-dependent formation
of a tetrameric gating ring spanning the intracellular region (IR)
of HCN. In the absence of cAMP, the IR cAMP-binding domain (CBD) mainly
samples its inactive conformation, resulting in steric clashes that
destabilize the IR tetramer. Although these clashes with the inactive
CBD are released through tetramer dissociation into monomers, functional
mutagenesis suggests that the apo IR is not fully monomeric. To investigate
the inhibitory nonmonomeric IR species, we performed molecular dynamics
simulations starting from “hybrid” structures that are
tetrameric but contain inactive apo-state CBD conformations. The ensemble
of simulated trajectories reveals that full dissociation of the tetramer
into monomers is not necessary to release the steric hindrance with
the inactive CBD. Specifically, we found that partial dissociation
of the tetramer into dimers is sufficient to accommodate four inactive
CBDs, while reduction of the quaternary symmetry of the nondissociated
tetramer from 4- to 2-fold permits accommodation of two inactive CBDs.
Our findings not only rationalize available electrophysiological,
fluorometry, and sedimentation equilibrium data, but also provide
unprecedented structural insight into previously elusive nonmonomeric
autoinhibitory HCN species
Role of Dimers in the cAMP-Dependent Activation of Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels
Hyperpolarization-activated
cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity
in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity
through the cAMP-induced formation of a tetrameric gating ring spanning
the intracellular region (IR) of HCN. Although evidence from confocal
patch-clamp fluorometry indicates that the cAMP-dependent gating of
HCN occurs through a dimer of dimers, the structural and dynamical
basis of cAMP allostery in HCN dimers has so far remained elusive.
Thus, here we examine how dimers influence IR structural dynamics,
and the role that such structural dynamics play in HCN allostery.
To this end, we performed molecular dynamics (MD) simulations of HCN4
IR dimers in their fully apo, fully holo, and partially cAMP-bound
states, resulting in a total simulated time of 1.2 μs. Comparative
analyses of these MD trajectories, as well as previous monomer and
tetramer simulations utilized as benchmarks for comparison, reveal
that dimers markedly sensitize the HCN IR to cAMP-modulated allostery.
Our results indicate that dimerization fine-tunes the IR dynamics
to enhance, relative to both monomers and tetramers, the allosteric
intra- and interprotomer coupling between the cAMP-binding domain
and tetramerization domain components of the IR. The resulting allosteric
model provides a viable rationalization of electrophysiological data
on the role of IR dimers in HCN activation
Role of Dynamics in the Autoinhibition and Activation of the Exchange Protein Directly Activated by Cyclic AMP (EPAC)
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Crystal structure of cGMP-dependent protein kinase Iβ cyclic nucleotide-binding-B domain : Rp-cGMPS complex reveals an apo-like, inactive conformation.
The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. Here, we determined the crystal structure of the PKG Iβ cyclic nucleotide-binding domain (PKG Iβ CNB-B), considered a 'gatekeeper' for cGMP activation, bound to Rp-cGMPS at 1.3 Å. Our structural and NMR data show that PKG Iβ CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalytic subunit-bound inhibited state of PKA RIα more closely than the apo or Rp-cAMPS-bound conformations. These results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B