scholarly journals Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14-3-3 ζ Protein

2021 ◽  
Author(s):  
Sreevidya T S ◽  
Somavally Dalvi ◽  
Prasanna Venkatraman ◽  
Satyavani Vemparala
Keyword(s):  
Electrostatic Interactions ◽  
Structural Changes ◽  
Structural Integrity ◽  
Peptide Binding ◽  
Limited Proteolysis ◽  
Binding Pocket ◽  
Mutant Protein ◽  
Amino Acid Residues ◽  
Salt Bridges ◽  
Dynamics Simulations

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3ζ isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3ζ. These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

scholarly journals SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure

2019 ◽  
Vol 24 (9) ◽  
pp. 928-938 ◽  
Author(s):  
Luca Palazzolo ◽  
Chiara Paravicini ◽  
Tommaso Laurenzi ◽  
Sara Adobati ◽  
Simona Saporiti ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Amino Acid ◽  
Molecular Dynamics Simulations ◽  
Electrostatic Interactions ◽  
Amino Acid Residues ◽  
Dibasic Amino Acid ◽  
Novel Target ◽  
Function Relationship ◽  
Dynamics Simulations

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane α-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure–function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport.

scholarly journals Extracellular Cation Binding Pocket Is Essential For Ion Conduction Of OsHKT2;2

2018 ◽  
Author(s):  
Janin Riedelsberger ◽  
Ariela Vergara-Jaque ◽  
Miguel Piñeros ◽  
Ingo Dreyer ◽  
Wendy González
Keyword(s):  
Protein Interactions ◽  
Mutual Effect ◽  
Homology Model ◽  
Binding Pocket ◽  
Cation Binding ◽  
Ion Binding ◽  
Salt Bridges ◽  
Extracellular Medium ◽  
Dynamics Simulations ◽  
Sodium And Potassium

AbstractHKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity, and performed molecular dynamics simulations in the presence of sodium and potassium ions. By analyzing ion-protein interactions, we identified a cation binding pocket on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion binding pocket and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results.

scholarly journals Dynamic Control of Ca2+ Binding in the C2 Domains of Synaptotagmin 1

2018 ◽  
Author(s):  
Patrick J. Rock ◽  
Austin G. Meyer ◽  
Chantell S. Evans ◽  
Edwin R. Chapman ◽  
R. Bryan Sutton
Keyword(s):  
Structural Changes ◽  
Dynamic Control ◽  
Point Mutations ◽  
Binding Pocket ◽  
Snare Complex ◽  
C2 Domains ◽  
Domain Interactions ◽  
Synaptotagmin 1 ◽  
Binding Pockets ◽  
Dynamics Simulations

AbstractSynaptotagmin senses fluctuations in the Ca2+ environment of neurons near active zones and transduces a signal to the SNARE complex to initiate exocytosis at the presynaptic terminus. The 3D structures of the two tandem C2 domains of synaptotagmin have been determined to high resolution; however, it is currently unclear how each domain dynamically interacts with Ca2+ at the atomic level. To study the mechanistic consequences of the lethal mutations at the AD3 locus, we introduced tyrosine to asparagine point mutations in both the C2A and C2B domains of synaptotagmin 1, and we have constructed a model that describes the relationship between Ca2+ -binding and the structural changes within each C2 domain. We show that the mobility of loop 3 in the Ca2+ binding pocket increases markedly in C2A, while the mobility of loop 1 changes in C2B with the AD3 mutation. This increase in loop mobility results in an increase in the average volume and variance of the Ca2+ -binding pockets of C2A and C2B. The volume of the unbound Ca2+ -binding pocket in C2A is usually restrained by intra-domain interactions between the tyrosine residue at the AD3 locus and residues on loop 3; however, the AD3 mutation decouples the restraint and results in a larger, more variable Ca2+ -binding pocket in C2A. C2B maintains a more compact Ca2+ -binding pocket; however, its volume also fluctuates significantly with the AD3 mutation. Changes in binding pocket volume that involve more variable Ca2+ binding loops would likely affect Ca2+ affinity in the neurons of the affected organism. Using molecular-dynamics simulations, we show that mutations at the AD3 locus alter the mobility of the Ca2+ -binding loops by removing a key stabilization mechanism that is normally present in C2 domains. The lack of loop stabilization results in a net increase in the volume of the Ca2+ -binding pocket and provides an explanation for the observed lethal phenotype.

scholarly journals Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

2021 ◽  
Vol 77 (5) ◽  
pp. 645-662
Author(s):  
Risako Tamura-Sakaguchi ◽  
Rie Aruga ◽  
Mika Hirose ◽  
Toru Ekimoto ◽  
Takuya Miyake ◽  
...  
Keyword(s):  
Complex Formation ◽  
Structure Determination ◽  
Structural Changes ◽  
Insertion Site ◽  
3D Structure ◽  
Binding Pocket ◽  
Crystallographic Analysis ◽  
Stable Complex ◽  
X Ray Crystallography ◽  
Dynamics Simulations

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

scholarly journals Polymorphisms at Amino Acid Residues 141 and 154 Influence Conformational Variation in Ovine PrP

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Sujeong Yang ◽  
Alana M. Thackray ◽  
Lee Hopkins ◽  
Tom P. Monie ◽  
David F. Burke ◽  
...  
Keyword(s):  
Amino Acid ◽  
Structural Changes ◽  
Classical Scrapie ◽  
Side Chain ◽  
Genotypic Differences ◽  
Amino Acid Side Chain ◽  
Atypical Scrapie ◽  
Amino Acid Residues ◽  
Structural Environment ◽  
Dynamics Simulations

Polymorphisms in ovine PrP at amino acid residues 141 and 154 are associated with susceptibility to ovine prion disease: Leu141Arg154 with classical scrapie and Phe141Arg154 and Leu141His154 with atypical scrapie. Classical scrapie is naturally transmissible between sheep, whereas this may not be the case with atypical scrapie. Critical amino acid residues will determine the range or stability of structural changes within the ovine prion protein or its functional interaction with potential cofactors, during conversion of PrPC to PrPSc in these different forms of scrapie disease. Here we computationally identified that regions of ovine PrP, including those near amino acid residues 141 and 154, displayed more conservation than expected based on local structural environment. Molecular dynamics simulations showed these conserved regions of ovine PrP displayed genotypic differences in conformational repertoire and amino acid side-chain interactions. Significantly, Leu141Arg154 PrP adopted an extended beta sheet arrangement in the N-terminal palindromic region more frequently than the Phe141Arg154 and Leu141His154 variants. We supported these computational observations experimentally using circular dichroism spectroscopy and immunobiochemical studies on ovine recombinant PrP. Collectively, our observations show amino acid residues 141 and 154 influence secondary structure and conformational change in ovine PrP that may correlate with different forms of scrapie.

scholarly journals Strong reduction of the chain rigidity of hyaluronan by selective binding of Ca2+ ions

2020 ◽  
Author(s):  
G. Giubertoni ◽  
A. Pérez de Alba Ortíz ◽  
F. Bano ◽  
X. Zhang ◽  
R.J. Linhardt ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Electrostatic Interactions ◽  
Structural Changes ◽  
Polymer Chains ◽  
Single Chain ◽  
Molecular Hydrogen Bond ◽  
Dynamics Simulations ◽  
Chain Force ◽  
Molecular Picture

ABSTRACTThe biological functions of natural polyelectrolytes are strongly influenced by the presence of ions, which bind to the polymer chains and thereby modify their properties. Although the biological impact of such modifications is well-recognized, a detailed molecular picture of the binding process and of the mechanisms that drive the subsequent structural changes in the polymer is lacking. Here, we study the molecular mechanism of the condensation of calcium, a divalent cation, on hyaluronan, a ubiquitous polymer in human tissues. By combining two-dimensional infrared spectroscopy experiments with molecular dynamics simulations, we find that calcium specifically binds to hyaluronan at millimolar concentrations. Because of its large size and charge, the calcium cation can bind simultaneously to the negatively charged carboxylate group and the amide group of adjacent saccharide units. Molecular dynamics simulations and single-chain force spectroscopy measurements provide evidence that the binding of the calcium ions weakens the intra-molecular hydrogen-bond network of hyaluronan, increasing the flexibility of the polymer chain. We also observe that the binding of calcium to hyaluronan saturates at a maximum binding fraction of ~10-15 mol %. This saturation indicates that the binding of Ca2+ strongly reduces the probability of subsequent binding of Ca2+ at neighboring binding sites, possibly as a result of enhanced conformational fluctuations and/or electrostatic repulsion effects. Our findings provide a detailed molecular picture of ion condensation, and reveal the severe effect of a few, selective and localized electrostatic interactions on the rigidity of a polyelectrolyte chain.TOC

scholarly journals Binding of Azobenzene and p-Diaminoazobenzene to the Human Voltage-Gated Sodium Channel NaV1.4

2020 ◽  
Author(s):  
Vito F. Palmisano ◽  
Carlos Gómez-Rodellar ◽  
Hannah Pollak ◽  
Gustavo Cárdenas ◽  
Ben Corry ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Internal Cavity ◽  
Amino Groups ◽  
Voltage Gated ◽  
Accelerated Molecular Dynamics ◽  
Binding Pockets ◽  
Voltage Gated Ion Channels ◽  
Dynamics Simulations

The activity of voltage-gated ion channels can be controlled by the binding of photoswitches inside their internal cavity and subsequent light irradiation. We investigated the binding of azobenzene and p-diaminoazobenzene to the human Na<sub>V</sub>1.4 channel in the inactivated state by means of Gaussian accelerated molecular dynamics simulations and free-energy computations. Three stable binding pockets were identified for each of the two photoswitches. In all the cases, the binding is controlled by the balance between the favorable hydrophobic interactions of the ligands with the nonpolar residues of the protein and the unfavorable polar solvation energy. In addition, electrostatic interactions between the ligand and the polar aminoacids are also relevant for p-diaminoazobenzene due to the presence of the amino groups on the benzene moieties. These groups participate in hydrogen bonding in the most favorable binding pocket and in long-range electrostatic interactions in the other pockets. The thermodinamically preferred binding sites found for both photoswitches are close to the selectivity filter of the channel. Therefore, it is very likely that the binding of these ligands will induce alterations in the ion conduction through the channel.

scholarly journals Hidden electrostatic basis of dynamic allostery in a PDZ domain

2017 ◽  
Vol 114 (29) ◽  
pp. E5825-E5834 ◽  
Author(s):  
Amit Kumawat ◽  
Suman Chakrabarty
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Pdz Domain ◽  
Allosteric Modulation ◽  
Side Chain ◽  
Salt Bridges ◽  
Population Shift ◽  
Domain Protein ◽  
Dynamics Simulations

Allosteric effect implies ligand binding at one site leading to structural and/or dynamical changes at a distant site. PDZ domains are classic examples of dynamic allostery without conformational changes, where distal side-chain dynamics is modulated on ligand binding and the origin has been attributed to entropic effects. In this work, we unearth the energetic basis of the observed dynamic allostery in a PDZ3 domain protein using molecular dynamics simulations. We demonstrate that electrostatic interaction provides a highly sensitive yardstick to probe the allosteric modulation in contrast to the traditionally used structure-based parameters. There is a significant population shift in the hydrogen-bonded network and salt bridges involving side chains on ligand binding. The ligand creates a local energetic perturbation that propagates in the form of dominolike changes in interresidue interaction pattern. There are significant changes in the nature of specific interactions (nonpolar/polar) between interresidue contacts and accompanied side-chain reorientations that drive the major redistribution of energy. Interestingly, this internal redistribution and rewiring of side-chain interactions led to large cancellations resulting in small change in the overall enthalpy of the protein, thus making it difficult to detect experimentally. In contrast to the prevailing focus on the entropic or dynamic effects, we show that the internal redistribution and population shift in specific electrostatic interactions drive the allosteric modulation in the PDZ3 domain protein.

scholarly journals The Structural Changes in the Signaling Mechanism of Bacteriophytochromes in Solution Revealed by a Multiscale Computational Investigation

2021 ◽  
Author(s):  
Veronica Macaluso ◽  
Giacomo Salvadori ◽  
Lorenzo Cupellini ◽  
Benedetta Mennucci
Keyword(s):  
Structural Changes ◽  
Red Light ◽  
Underlying Mechanism ◽  
Binding Pocket ◽  
Spectroscopic Properties ◽  
Signaling Mechanism ◽  
Structure Relaxation ◽  
Light Sensing ◽  
Absorption Of Light ◽  
Dynamics Simulations

<pre>Phytochromes are red-light sensing proteins, with important light-regulatory roles in different organisms, which are capturing an increasing interest in bioimaging and optogenetics. Upon absorption of light by the embedded bilin chromophore, they undergo structural changes that extend from the chomophore to the protein and finally drive the biological function. Up to now, the underlying mechanism still has to be characterized fully. </pre> <pre>Here we investigate the Pfr activated form of a bacterial phytochrome, by combining extensive Molecular Dynamics simulations with a polarizable QM/MM description of the spectroscopic properties, revealing a large structure relaxation in solution, compared to the crystal structure, both in the chromophore-binding pocket and in the overall structure of the phytochrome. Our results indicate that the final opening of the dimeric structure is preceded by an important internal reorganization of the phytochrome specific (PHY) domain involving a bend of the helical spine connecting the PHY domain with the chromophore-binding domain, opening the way to a new understanding of the activation pathway. </pre>

Sign in / Sign up

Export Citation Format

Share Document