Imperial College London

DrAlfonsoDe Simone

Faculty of Natural SciencesDepartment of Life Sciences

Reader in Structural Biology
 
 
 
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Contact

 

+44 (0)20 7594 3052a.de-simon Website

 
 
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Location

 

603Sir Ernst Chain BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

77 results found

Heller GT, Aprile FA, Bonomi M, Camilloni C, De Simone A, Vendruscolo Met al., 2017, Sequence Specificity in the Entropy-Driven Binding of a Small Molecule and a Disordered Peptide., J Mol Biol, Vol: 429, Pages: 2772-2779

Approximately one-third of the human proteome is made up of proteins that are entirely disordered or that contain extended disordered regions. Although these disordered proteins are closely linked with many major diseases, their binding mechanisms with small molecules remain poorly understood, and a major concern is whether their specificity can be sufficient for drug development. Here, by studying the interaction of a small molecule and a disordered peptide from the oncogene protein c-Myc, we describe a "specific-diffuse" binding mechanism that exhibits sequence specificity despite being of entropic nature. By combining NMR spectroscopy, biophysical measurements, statistical inference, and molecular simulations, we provide a quantitative measure of such sequence specificity and compare it to the case of the interaction of urea, which is diffuse but not specific. To investigate whether this type of binding can generally modify intermolecular interactions, we show that it leads to an inhibition of the aggregation of the peptide. These results suggest that the binding mechanism that we report may create novel opportunities to discover drugs that target disordered proteins in their monomeric states in a specific manner.

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Ruggiero A, Squeglia F, Romano M, Vitagliano L, De Simone A, Berisio Ret al., 2017, Structure and dynamics of the multi-domain resuscitation promoting factor RpfB from Mycobacterium tuberculosis, JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS, Vol: 35, Pages: 1322-1330, ISSN: 0739-1102

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Toto A, Bonetti D, De Simone A, Gianni Set al., 2017, Understanding the mechanism of binding between Gab2 and the C terminal SH3 domain from Grb2., Oncotarget

Gab2 is a large disordered protein that regulates several cellular signalling pathways and is overexpressed in different forms of cancer. Because of its disordered nature, a detailed characterization of the mechanisms of recognition between Gab2 and its physiological partners is particularly difficult. Here we provide a detailed kinetic characterization of the binding reaction between Gab2 and the C-terminal SH3 domain of the growth factor receptor-bound protein 2 (Grb2). We demonstrate that Gab2 folds upon binding following an induced fit type mechanism, whereby recognition is characterized by the formation of an intermediate, in which Gab2 is primarily disordered. In this scenario, folding of Gab2 into the bound conformation occurs only after binding. However, an alanine scanning of the proline residues of Gab2 suggests that the intermediate contains some degree of native-like structure, which might play a role for the recognition event to take place. The results, which represent a fundamental step forward in the understanding of this functional protein-protein interaction, are discussed on the light of previous structural works on these proteins.

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Fusco G, De Simone A, Arosio P, Vendruscolo M, Veglia G, Dobson CMet al., 2016, Structural Ensembles of Membrane-bound alpha-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity, SCIENTIFIC REPORTS, Vol: 6, ISSN: 2045-2322

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Fusco G, Pape T, Stephens AD, Mahou P, Costa AR, Kaminski CF, Schierle GSK, Vendruscolo M, Veglia G, Dobson CM, De Simone Aet al., 2016, Structural basis of synaptic vesicle assembly promoted by alpha-synuclein, NATURE COMMUNICATIONS, Vol: 7, ISSN: 2041-1723

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Ruggiero A, Squeglia F, Romano M, Vitagliano L, De Simone A, Berisio Ret al., 2016, The structure of Resuscitation promoting factor B from M. tuberculosis reveals unexpected ubiquitin-like domains, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, Vol: 1860, Pages: 445-451, ISSN: 0304-4165

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Sanfelice D, Sanz-Hernández M, de Simone A, Bullard B, Pastore Aet al., 2016, Toward Understanding the Molecular Bases of Stretch Activation: A STRUCTURAL COMPARISON OF THE TWO TROPONIN C ISOFORMS OF LETHOCERUS., J Biol Chem, Vol: 291, Pages: 16090-16099

Muscles are usually activated by calcium binding to the calcium sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle activation is also produced by mechanical stress. Little is known about the molecular bases of this calcium-independent activation. In Lethocerus, a giant water bug often used as a model system because of its large muscle fibers, there are two troponin-C isoforms, called F1 and F2, that have distinct roles in activating the muscle. It has been suggested that this can be explained either by differences in structural features or by differences in the interactions with other proteins. Here we have compared the structural and dynamic properties of the two proteins and shown how they differ. We have also mapped the interactions of the F2 isoform with peptides spanning the sequence of its natural partner, troponin-I. Our data have allowed us to build a model of the troponin complex and may eventually help in understanding the specialized function of the F1 and F2 isoforms and the molecular mechanism of stretch activation.

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Sanz-Hernandez M, Vostrikov VV, Veglia G, De Simone Aet al., 2016, Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins, SCIENTIFIC REPORTS, Vol: 6, ISSN: 2045-2322

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Taylor JD, Hawthorne WJ, Lo J, Dear A, Jain N, Meisl G, Andreasen M, Fletcher C, Koch M, Darvill N, Scull N, Escalera-Maurer A, Sefer L, Wenman R, Lambert S, Jean J, Xu Y, Turner B, Kazarian SG, Chapman MR, Bubeck D, de Simone A, Knowles TPJ, Matthews SJet al., 2016, Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones, SCIENTIFIC REPORTS, Vol: 6, ISSN: 2045-2322

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De Simone A, Aprile FA, Dhulesia A, Dobson CM, Vendruscolo Met al., 2015, Structure of a low-population intermediate state in the release of an enzyme product., Elife, Vol: 4

Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product.

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Granata D, Baftizadeh F, Habchi J, Galvagnion C, De Simone A, Camilloni C, Laio A, Vendruscolo Met al., 2015, The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments., Sci Rep, Vol: 5

The free energy landscape theory has been very successful in rationalizing the folding behaviour of globular proteins, as this representation provides intuitive information on the number of states involved in the folding process, their populations and pathways of interconversion. We extend here this formalism to the case of the Aβ40 peptide, a 40-residue intrinsically disordered protein fragment associated with Alzheimer's disease. By using an advanced sampling technique that enables free energy calculations to reach convergence also in the case of highly disordered states of proteins, we provide a precise structural characterization of the free energy landscape of this peptide. We find that such landscape has inverted features with respect to those typical of folded proteins. While the global free energy minimum consists of highly disordered structures, higher free energy regions correspond to a large variety of transiently structured conformations with secondary structure elements arranged in several different manners, and are not separated from each other by sizeable free energy barriers. From this peculiar structure of the free energy landscape we predict that this peptide should become more structured and not only more compact, with increasing temperatures, and we show that this is the case through a series of biophysical measurements.

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Kukic P, Alvin Leung HT, Bemporad F, Aprile FA, Kumita JR, De Simone A, Camilloni C, Vendruscolo Met al., 2015, Structure and dynamics of the integrin LFA-1 I-domain in the inactive state underlie its inside-out/outside-in signaling and allosteric mechanisms., Structure, Vol: 23, Pages: 745-753

Lymphocyte function-associated antigen 1 (LFA-1) is an integrin that transmits information in two directions across the plasma membrane of leukocytes, in so-called outside-in and inside-out signaling mechanisms. To investigate the structural basis of these mechanisms, we studied the conformational space of the apo I-domain using replica-averaged metadynamics simulations in combination with nuclear magnetic resonance chemical shifts. We thus obtained a free energy landscape that reveals the existence of three conformational substates of this domain. The three substates include conformations similar to existing crystallographic structures of the low-affinity I-domain, the inactive I-domain with an allosteric antagonist inhibitor bound underneath α helix 7, and an intermediate affinity state of the I-domain. The multiple substates were validated with residual dipolar coupling measurements. These results suggest that the presence of three substates in the apo I-domain enables the precise regulation of the binding process that is essential for the physiological function of LFA-1.

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Maione V, Ruggiero A, Russo L, De Simone A, Pedone PV, Malgieri G, Berisio R, Isernia Cet al., 2015, NMR Structure and Dynamics of the Resuscitation Promoting Factor RpfC Catalytic Domain., PLoS One, Vol: 10

Mycobacterium tuberculosis latent infection is maintained for years with no clinical symptoms and no adverse effects for the host. The mechanism through which dormant M. tuberculosis resuscitates and enters the cell cycle leading to tuberculosis is attracting much interest. The RPF family of proteins has been found to be responsible for bacteria resuscitation and normal proliferation. This family of proteins in M. tuberculosis is composed by five homologues (named RpfA-E) and understanding their conformational, structural and functional peculiarities is crucial to the design of therapeutic strategies.Therefore, we report the structural and dynamics characterization of the catalytic domain of RpfC from M. tubercolosis by combining Nuclear Magnetic Resonance, Circular Dichroism and Molecular Dynamics data. We also show how the formation of a disulfide bridge, highly conserved among the homologues, is likely to modulate the shape of the RpfC hydrophobic catalytic cleft. This might result in a protein function regulation via a "conformational editing" through a disulfide bond formation.

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Nasica-Labouze J, Nguyen PH, Sterpone F, Berthoumieu O, Buchete N-V, Cote S, De Simone A, Doig AJ, Faller P, Garcia A, Laio A, Li MS, Melchionna S, Mousseau N, Mu Y, Paravastu A, Pasquali S, Rosenman DJ, Strodel B, Tarus B, Viles JH, Zhang T, Wang C, Derreumaux Pet al., 2015, Amyloid beta Protein and Alzheimer's Disease: When Computer Simulations Complement Experimental Studies, CHEMICAL REVIEWS, Vol: 115, Pages: 3518-3563, ISSN: 0009-2665

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Newby FN, De Simone A, Yagi-Utsumi M, Salvatella X, Dobson CM, Vendruscolo Met al., 2015, Structure-Free Validation of Residual Dipolar Coupling and Paramagnetic Relaxation Enhancement Measurements of Disordered Proteins., Biochemistry, Vol: 54, Pages: 6876-6886

Residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs) have emerged as valuable parameters for defining the structures and dynamics of disordered proteins by nuclear magnetic resonance (NMR) spectroscopy. Procedures for their measurement, however, may lead to conformational perturbations because of the presence of the alignment media necessary for recording RDCs, or of the paramagnetic groups that must be introduced for measuring PREs. We discuss here experimental methods for quantifying these effects by considering the case of the 40-residue isoform of the amyloid β peptide (Aβ40), which is associated with Alzheimer's disease. By conducting RDC measurements over a range of concentrations of certain alignment media, we show that perturbations arising from transient binding of Aβ40 can be characterized, allowing appropriate corrections to be made. In addition, by using NMR experiments sensitive to long-range interactions, we show that it is possible to identify relatively nonperturbing sites for attaching nitroxide radicals for PRE measurements. Thus, minimizing the conformational perturbations introduced by RDC and PRE measurements should facilitate their use for the rigorous determination of the conformational properties of disordered proteins.

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De Simone A, 2014, Reply to: "Only kinetics can prove conformational selection"., Biophys J, Vol: 107, Pages: 1999-2000

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De Simone A, Mote KR, Veglia G, 2014, Structural dynamics and conformational equilibria of SERCA regulatory proteins in membranes by solid-state NMR restrained simulations., Biophys J, Vol: 106, Pages: 2566-2576

Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes.

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Fusco D, Headd JJ, De Simone A, Wang J, Charbonneau Pet al., 2014, Characterizing protein crystal contacts and their role in crystallization: rubredoxin as a case study, SOFT MATTER, Vol: 10, Pages: 290-302, ISSN: 1744-683X

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Fusco G, De Simone A, Gopinath T, Vostrikov V, Vendruscolo M, Dobson CM, Veglia Get al., 2014, Direct observation of the three regions in alpha-synuclein that determine its membrane-bound behaviour, NATURE COMMUNICATIONS, Vol: 5, ISSN: 2041-1723

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Krieger JM, Fusco G, Lewitzky M, Simister PC, Marchant J, Camilloni C, Feller SM, De Simone Aet al., 2014, Conformational Recognition of an Intrinsically Disordered Protein, BIOPHYSICAL JOURNAL, Vol: 106, Pages: 1771-1779, ISSN: 0006-3495

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Leung HTA, Kukic P, Camilloni C, Bemporad F, De Simone A, Aprile FA, Kumita JR, Vendruscolo Met al., 2014, NMR characterization of the conformational fluctuations of the human lymphocyte function-associated antigen-1 I-domain., Protein Sci, Vol: 23, Pages: 1596-1606

Lymphocyte function-associated antigen-1 (LFA-1) is an integrin protein that transmits information across the plasma membrane through the so-called inside-out and outside-in signaling mechanisms. To investigate these mechanisms, we carried out an NMR analysis of the dynamics of the LFA-1 I-domain, which has enabled us to characterize the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model-free analysis. We then extended this analysis to the millisecond timescale motions by measuring (15) N-(1) H residual dipolar couplings of the backbone amide groups. We analyzed these results in the context of the three major conformational states of the I-domain using their corresponding X-ray crystallographic structures. Our results highlight the importance of the low-frequency motions of the LFA-1 I-domain in the inactive apo-state. We found in particular that α-helix 7 is in a position in the apo-closed state that cannot be fully described by any of the existing X-ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA-1 I-domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of α-helix 7 that is required for the activation of LFA-1.

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Montalvao R, Camilloni C, De Simone A, Vendruscolo Met al., 2014, New opportunities for tensor-free calculations of residual dipolar couplings for the study of protein dynamics., J Biomol NMR, Vol: 58, Pages: 233-238

Residual dipolar couplings (RDCs) can provide exquisitely detailed information about the structure and dynamics of proteins. It is challenging, however, to extract such information from RDC measurements in conformationally heterogeneous states of proteins because of the complex relationship between RDCs and protein structures. To obtain new insights into this problem, we discuss methods of calculating the RDCs that do not require the definition of an alignment tensor. These methods can help in particular in the search of effective ways to use RDCs to characterise disordered or partially disordered states of proteins.

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Sanfelice D, De Simone A, Cavalli A, Faggiano S, Vendruscolo M, Pastore Aet al., 2014, Characterization of the conformational fluctuations in the Josephin domain of ataxin-3., Biophys J, Vol: 107, Pages: 2932-2940

As for a variety of other molecular recognition processes, conformational fluctuations play an important role in the cleavage of polyubiquitin chains by the Josephin domain of ataxin-3. The interaction between Josephin and ubiquitin appears to be mediated by the motions of α-helical hairpin that is unusual among deubiquitinating enzymes. Here, we characterized the conformational fluctuations of the helical hairpin by incorporating NMR measurements as replica-averaged restraints in molecular dynamics simulations, and by validating the results by small-angle x-ray scattering measurements. This approach allowed us to define the extent of the helical hairpin motions and suggest a role of such motions in the recognition of ubiquitin.

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Squeglia F, Bachert B, De Simone A, Lukomski S, Berisio Ret al., 2014, The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41., J Biol Chem, Vol: 289, Pages: 5122-5133

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

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Balasco N, Esposito L, De Simone A, Vitagliano Let al., 2013, Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms., Protein Sci, Vol: 22, Pages: 1016-1023

It has been recently discovered that the connection of secondary structure elements (ββ-unit, βα- and αβ-units) in proteins follows quite stringent principles regarding the chirality and the orientation of the structural units (Koga et al., Nature 2012;491:222-227). By exploiting these rules, a number of protein scaffolds endowed with a remarkable thermal stability have been designed (Koga et al., Nature 2012;491:222-227). By using structural databases of proteins isolated from either mesophilic or thermophilic organisms, we here investigate the influence of supersecondary associations on the thermal stability of natural proteins. Our results suggest that β-hairpins of proteins from thermophilic organisms are very frequently characterized by shortenings of the loops. Interestingly, this shortening leads to states that display a very strong preference for the most common connectivity of the strands observed in native protein hairpins. The abundance of selective states in these proteins suggests that they may achieve a high stability by adopting a strategy aimed to reduce the possible conformations of the unfolded ensemble. In this scenario, our data indicate that the shortening is effective if it increases the adherence to these rules. We also show that this mechanism may operate in the stabilization of well-known protein folds (thioredoxin and RNase A). These findings suggest that future investigations aimed at defining mechanism of protein stabilization should also consider these effects.

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Biedermann F, Vendruscolo M, Scherman OA, De Simone A, Nau WMet al., 2013, Cucurbit[8]uril and blue-box: high-energy water release overwhelms electrostatic interactions., J Am Chem Soc, Vol: 135, Pages: 14879-14888

The design of high-affinity and analyte-selective receptors operating in aqueous solutions is an outstanding problem in supramolecular chemistry. Directing the focus toward the unique properties of water, we present here a new strategy toward this goal and support it by molecular dynamics simulations and calorimetric measurements. We illustrate the procedure in the case of self-assembled 1:1 complexes of the rigid macrocycle cucurbit[8]uril (CB8) and dicationic auxiliary guests (AG). These CB8•AG complexes contain residual water molecules whose conformational space and hydrogen-bond formation ability is restricted by the geometrically confined and hydrophobic cavity of the receptor. We show that upon inclusion of an analyte to form a 1:1:1 CB8•AG•analyte complex, these "high-energy" cavity water molecules are released to the aqueous bulk, providing a strong enthalpic driving force to the association, and resulting in binding constants of up to 10(6) M(-1) for aromatic analytes. This binding model is supported by the measurements of large solvent and solvent isotope effects. The selectivity of the CB8•AG receptor can be modified or even switched toward small aliphatic analytes by a rational choice of the auxiliary guest, demonstrating the tunable recognition features of such self-assembled receptors. Furthermore, by comparison of the results to those for the extensively studied macrocyclic host cyclobis(paraquat-p-phenylene)--the so-called "blue-box"--it is shown that in aqueous solution the release of "high-energy" water molecules from the CB8•AG cavity can be more favorable than the use of direct host-guest interactions.

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Borkar AN, De Simone A, Montalvao RW, Vendruscolo Met al., 2013, A method of determining RNA conformational ensembles using structure-based calculations of residual dipolar couplings., J Chem Phys, Vol: 138

We describe a method of determining the conformational fluctuations of RNA based on the incorporation of nuclear magnetic resonance (NMR) residual dipolar couplings (RDCs) as replica-averaged structural restraints in molecular dynamics simulations. In this approach, the alignment tensor required to calculate the RDCs corresponding to a given conformation is estimated from its shape, and multiple replicas of the RNA molecule are simulated simultaneously to reproduce in silico the ensemble-averaging procedure performed in the NMR measurements. We provide initial evidence that with this approach it is possible to determine accurately structural ensembles representing the conformational fluctuations of RNA by applying the reference ensemble test to the trans-activation response element of the human immunodeficiency virus type 1.

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Cantisani M, Falanga A, Incoronato N, Russo L, De Simone A, Morelli G, Berisio R, Galdiero M, Galdiero Set al., 2013, Conformational modifications of gB from herpes simplex virus type 1 analyzed by synthetic peptides., J Med Chem, Vol: 56, Pages: 8366-8376

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. The crystallized trimeric glycoprotein gB of herpes simplex virus has been described as a postfusion conformation, and several studies prove that like other class III fusion proteins, gB undergoes a pH-dependent switch between the pre- and postfusion conformations. Using several biophysical techniques, we show that peptides corresponding to the long helix of the gB postfusion structure interfere with the membrane fusion event, likely hampering the conformational rearrangements from the pre- to the postfusion structures. Those peptides represent good candidates for further design of peptidomimetic antagonists capable of blocking the fusion process.

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De Simone A, Gustavsson M, Montalvao RW, Shi L, Veglia G, Vendruscolo Met al., 2013, Structures of the excited states of phospholamban and shifts in their populations upon phosphorylation., Biochemistry, Vol: 52, Pages: 6684-6694

Phospholamban is an integral membrane protein that controls the calcium balance in cardiac muscle cells. As the function and regulation of this protein require the active involvement of low populated states in equilibrium with the native state, it is of great interest to acquire structural information about them. In this work, we calculate the conformations and populations of the ground state and the three main excited states of phospholamban by incorporating nuclear magnetic resonance residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations. We then provide a description of the manner in which phosphorylation at Ser16 modulates the activity of the protein by increasing the sizes of the populations of its excited states. These results demonstrate that the approach that we describe provides a detailed characterization of the different states of phospholamban that determine the function and regulation of this membrane protein. We anticipate that the knowledge of conformational ensembles enable the design of new dominant negative mutants of phospholamban by modulating the relative populations of its conformational substates.

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De Simone A, Montalvao RW, Dobson CM, Vendruscolo Met al., 2013, Characterization of the interdomain motions in hen lysozyme using residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations., Biochemistry, Vol: 52, Pages: 6480-6486

Hen lysozyme is an enzyme characterized by the presence of two domains whose relative motions are involved in the mechanism of binding and release of the substrates. By using residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations, we characterize the breathing motions describing the interdomain fluctuations of this protein. We found that the ensemble of conformations that we determined spans the entire range of structures of hen lysozyme deposited in the Protein Data Bank, including both the free and bound states, suggesting that the thermal motions in the free state provide access to the structures populated upon binding. The approach that we present illustrates how the use of residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations makes it possible to explore conformational fluctuations of a relatively large amplitude in proteins.

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