Pockets as structural descriptors of EGFR kinase conformations

Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor, is one of the main tumor markers in different types of cancers. The kinase native state is mainly composed of two populations of conformers: active and inactive. Several sequence variations in EGFR kinase region promote the differential enrichment of conformers with higher activity. Some structural characteristics have been proposed to differentiate kinase conformations, but these considerations could lead to ambiguous classifications. We present a structural characterisation of EGFR kinase conformers, focused on active site pocket comparisons, and the mapping of known pathological sequence variations. A structural based clustering of this pocket accurately discriminates active from inactive, well-characterised conformations. Furthermore, this main pocket contains, or is in close contact with, ≈65% of cancer-related variation positions. Although the relevance of protein dynamics to explain biological function has been extensively recognised, the usage of the ensemble of conformations in dynamic equilibrium to represent the functional state of proteins and the importance of pockets, cavities and/or tunnels was often neglected in previous studies. These functional structures and the equilibrium between them could be structurally analysed in wild type as well as in sequence variants. Our results indicate that biologically important pockets, as well as their shape and dynamics, are central to understanding protein function in wild-type, polymorphic or disease-related variations.Fil: Hasenahuer, Marcia Anahí. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barletta Roldan, Patricio German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Alberti, Sebastián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Parisi, Gustavo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fornasari, Maria Silvina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Fatty acid and retinol-binding protein: Unusual protein conformational and cavity changes dictated by ligand fluctuations

Lipid-binding proteins (LBPs) are soluble proteins responsible for the uptake, transport, and storage of a large variety of hydrophobic lipophilic molecules including fatty acids, steroids, and other lipids in the cellular environment. Among the LBPs, fatty acid binding proteins (FABPs) present preferential binding affinities for long-chain fatty acids. While most of FABPs in vertebrates and invertebrates present similar β-barrel structures with ligands accommodated in their central cavity, parasitic nematode worms exhibit additional unusual α-helix rich fatty acid- and retinol-binding proteins (FAR). Herein, we report the comparison of extended molecular dynamics (MD) simulations performed on the ligand-free and palmitic acid-bond states of the Necator americanus FAR-1 (Na-FAR-1) with respect to other classical β-barrel FABPs. Principal component analysis (PCA) has been used to identify the different conformations adopted by each system during MD simulations. The α-helix fold encompasses a complex internal ligand-binding cavity with a remarkable conformational plasticity that allows reversible switching between distinct states in the holo-Na-FAR-1. The cavity can change up to one-third of its size affected by conformational changes of the protein-ligand complex. Besides, the ligand inside the cavity is not fixed but experiences large conformational changes between bent and stretched conformations. These changes in the ligand conformation follow changes in the cavity size dictated by the transient protein conformation. On the contrary, protein-ligand complex in β-barrel FABPs fluctuates around a unique conformation. The significantly more flexible holo-Na-FAR-1 ligand-cavity explains its larger ligand multiplicity respect to β-barrel FABPs.Fil: Barletta Roldan, Patricio German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Franchini, Gisela Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Córsico, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; ArgentinaFil: Fernández Alberti, Sebastián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Antibody-Antigen Binding Interface Analysis in the Big Data Era

Antibodies have become the Swiss Army tool for molecular biology and nanotechnology. Their outstanding ability to specifically recognise molecular antigens allows their use in many different applications from medicine to the industry. Moreover, the improvement of conventional structural biology techniques (e.g., X-ray, NMR) as well as the emergence of new ones (e.g., Cryo-EM), have permitted in the last years a notable increase of resolved antibody-antigen structures. This offers a unique opportunity to perform an exhaustive structural analysis of antibody-antigen interfaces by employing the large amount of data available nowadays. To leverage this factor, different geometric as well as chemical descriptors were evaluated to perform a comprehensive characterization.Fil: Reis, Pedro B. P. S.. Istituto Italiano Di Technologie; Italia. Universidade Nova de Lisboa; PortugalFil: Barletta Roldan, Patricio German. The Abdus Salam; Italia. The Abdus Salam. International Centre for Theoretical Physics; Italia. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gagliardi, Lucas. Istituto Italiano Di Technologie; ItaliaFil: Fortuna, Sara. Istituto Italiano Di Technologie; ItaliaFil: Soler, Miguel A.. Istituto Italiano Di Technologie; ItaliaFil: Rocchia, Walter. Istituto Italiano Di Technologie; Itali

Protein Fluctuations and Cavity Changes Relationship

Protein cavities and tunnels are critical for function. Ligand recognition and binding, transport, and enzyme catalysis require cavities rearrangements. Therefore, the flexibility of cavities should be guaranteed by protein vibrational dynamics. Molecular dynamics simulations provide a framework to explore conformational plasticity of protein cavities. Herein, we present a novel procedure to characterize the dynamics of protein cavities in terms of their volume gradient vector. For this purpose, we make use of algorithms for calculation of the cavity volume that result robust for numerical differentiations. Volume gradient vector is expressed in terms of principal component analysis obtained from equilibrated molecular dynamics simulations. We analyze contributions of principal component modes to the volume gradient vector according to their frequency and degree of delocalization. In all our test cases, we find that low frequency modes play a critical role together with minor contributions of high frequency modes. These modes involve concerted motions of significant fractions of the total residues lining the cavities. We make use of variations of the potential energy of a protein in the direction of the volume gradient vector as a measure of flexibility of the cavity. We show that proteins whose collective low frequency fluctuations contribute the most to changes of cavity volume exhibit more flexible cavities.Fil: Barletta Roldan, Patricio German. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Alberti, Sebastián. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Analysis of changes of cavity volumes in predefined directions of protein motions and cavity flexibility

Dynamics of protein cavities associated with protein fluctuations and conformationalplasticity is essential for their biological function. NMR ensembles, molecular dynamics (MD) simulations, and normal mode analysis (NMA) provide appropriate frameworks to explore functionally relevant protein dynamics and cavity changesrelationships. Within this context, we have recently developed analysis of null areas(ANA), an efficient method to calculate cavity volumes. ANA is based on a combination of algorithms that guarantees its robustness against numerical differentiations.This is a unique feature with respect to other methods. Herein, we present anupdated and improved version that expands it use to quantify changes in cavity features, like volume and flexibility, due to protein structural distortions performed onpredefined biologically relevant directions, for example, directions of largest contribution to protein fluctuations (principal component analysis [PCA modes]) obtained byMD simulations or ensembles of NMR structures, collective NMA modes or anyother direction of motion associated with specific conformational changes. A webpage has been developed where its facilities are explained in detail. First, we showthat ANA can be useful to explore gradual changes of cavity volume and flexibilityassociated with protein ligand binding. Secondly, we perform a comparison study ofthe extent of variability between protein backbone structural distortions, andchanges in cavity volumes and flexibilities evaluated for an ensemble of NMR activeand inactive conformers of the epidermal growth factor receptor structures. Finally,we compare changes in size and flexibility between sets of NMR structures for different homologous chains of dynein.Fil: Barletta Roldan, Patricio German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barletta, Matias. Lionix Evolve; Costa RicaFil: Saldaño, Tadeo Enrique. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Alberti, Sebastián. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Correction: Pockets as structural descriptors of EGFR kinase conformations.

[This corrects the article DOI: 10.1371/journal.pone.0189147.]

Dynamics fingerprints of active conformers of epidermal growth factor receptor kinase

Epidermal growth factor receptor (EGFR) is a prototypical cell-surface receptor that plays a key role in the regulation of cellular signaling, proliferation and differentiation. Mutations of its kinase domain have been associated with the development of a variety of cancers and, therefore, it has been the target of drug design. Single amino acid substitutions (SASs) in this domain have been proven to alter the equilibrium of pre-existing conformer populations. Despite the advances in structural descriptions of its so-called active and inactive conformations, the associated dynamics aspects that characterize them have not been thoroughly studied yet. As the dynamic behaviors and molecular motions of proteins are important for a complete understanding of their structure–function relationships we present a novel procedure, using (or based on) normal mode analysis, to identify the collective dynamics shared among different conformers in EGFR kinase. The method allows the comparison of patterns of low-frequency vibrational modes defining representative directions of motions. Our procedure is able to emphasize the main similarities and differences between the collective dynamics of different conformers. In the case of EGFR kinase, two representative directions of motions have been found as dynamics fingerprints of the active conformers. Protein motion along both directions reveals to have a significant impact on the cavity volume of the main pocket of the active site. Otherwise, the inactive conformers exhibit a more heterogeneous distribution of collective motions.Fil: Barletta Roldan, Patricio German. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Hasenahuer, Marcia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Fornasari, Maria Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Parisi, Gustavo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Fernández Alberti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin

Intrinsically Disordered Region Modulates Ligand Binding in Glutaredoxin 1 from Trypanosoma Brucei

Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron–sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron–sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.Fil: Balatti, Galo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Barletta Roldan, Patricio German. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Parisi, Gustavo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Tosatto, Silvio. C. E.. Università di Padova; ItaliaFil: Bellanda, Massimo. Università di Padova; ItaliaFil: Fernández Alberti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin

Pockets as structural descriptors of EGFR kinase conformations

<div><p>Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor, is one of the main tumor markers in different types of cancers. The kinase native state is mainly composed of two populations of conformers: <i>active</i> and <i>inactive</i>. Several sequence variations in EGFR kinase region promote the differential enrichment of conformers with higher activity. Some structural characteristics have been proposed to differentiate kinase conformations, but these considerations could lead to ambiguous classifications. We present a structural characterisation of EGFR kinase conformers, focused on active site pocket comparisons, and the mapping of known pathological sequence variations. A structural based clustering of this pocket accurately discriminates active from inactive, well-characterised conformations. Furthermore, this main pocket contains, or is in close contact with, ≈65% of cancer-related variation positions. Although the relevance of protein dynamics to explain biological function has been extensively recognised, the usage of the ensemble of conformations in dynamic equilibrium to represent the functional state of proteins and the importance of pockets, cavities and/or tunnels was often neglected in previous studies. These functional structures and the equilibrium between them could be structurally analysed in wild type as well as in sequence variants. Our results indicate that biologically important pockets, as well as their shape and dynamics, are central to understanding protein function in wild-type, polymorphic or disease-related variations.</p></div

Positions belonging to and in close contact with the main pocket.

<p>Exon: shows the limits of each exon. Region: informs the conserved regions involved in catalytic control. Abbreviations: CS: catalytic spine; ADi, SDi: assymetric/symmetric dimer interface; RS: regulatory spine; GK: gatekeeper. Position, list number and residue for each position in the main pocket and close contact. P/C: depicts whether a position is considered as part of the main pocket (P) or in close contact (C). Position and P/C rows are coloured, according to the considerations in P/C, for those sites with defined coordinates in all the structures used in the present work. Those in white correspond to positions that are found as missing in at least one of these structures.</p