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

Modeling the effect of pathogenic mutations on the conformational landscape of protein kinases

Most proteins assume different conformations to perform their cellular functions. This conformational dynamics is physiologically regulated by binding events and post-translational modifications, but can also be affected by pathogenic mutations. Atomistic molecular dynamics simulations complemented by enhanced sampling approaches are increasingly used to probe the effect of mutations on the conformational dynamics and on the underlying conformational free energy landscape of proteins. In this short review we discuss recent successful examples of simulations used to understand the molecular mechanism underlying the deregulation of physiological conformational dynamics due to non-synonymous single point mutations. Our examples are mostly drawn from the protein kinase family

GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding

Conserved microbe-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) act as danger signals to activate the plant immune response. These molecules are recognized by surface receptors that are referred to as pattern recognition receptors. Oligogalacturonides (OGs), DAMPs released from the plant cell wall homogalacturonan, have also been proposed to act as local signals in the response to wounding. The Arabidopsis Wall-Associated Kinase 1 (WAK1), a receptor of OGs, has been described to form a complex with a cytoplasmic plasma membrane-localized kinase-associated protein phosphatase (KAPP) and a glycine-rich protein (GRP-3) that we find localized mainly in the cell wall and, in a small part, on the plasma membrane. By using Arabidopsis plants overexpressing WAK1, and both grp-3 and kapp null insertional mutant and overexpressing plants, we demonstrate a positive function of WAK1 and a negative function of GRP-3 and KAPP in the OG-triggered expression of defence genes and the production of an oxidative burst. The three proteins also affect the local response to wounding and the basal resistance against the necrotrophic pathogen Botrytis cinerea. GRP-3 and KAPP are likely to function in the phasing out of the plant immune response

Allostery in Its Many Disguises: From Theory to Applications.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts

Coevolved mutations reveal distinct architectures for two core proteins in the bacterial flagellar motor

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.JK was supported by Medical Research Council grant U117581331. SK was supported by seed funds from Lahore University of Managment Sciences (LUMS) and the Molecular Biology Consortium

Integrating atomistic molecular dynamics simulations, experiments, and network analysis to study protein dynamics:strength in unity

In the last years, we have been observing remarkable improvements in the field of protein dynamics. Indeed, we can now study protein dynamics in atomistic details over several timescales with a rich portfolio of experimental and computational techniques. On one side, this provides us with the possibility to validate simulation methods and physical models against a broad range of experimental observables. On the other side, it also allows a complementary and comprehensive view on protein structure and dynamics. What is needed now is a better understanding of the link between the dynamic properties that we observe and the functional properties of these important cellular machines. To make progresses in this direction, we need to improve the physical models used to describe proteins and solvent in molecular dynamics, as well as to strengthen the integration of experiments and simulations to overcome their own limitations. Moreover, now that we have the means to study protein dynamics in great details, we need new tools to understand the information embedded in the protein ensembles and in their dynamic signature. With this aim in mind, we should enrich the current tools for analysis of biomolecular simulations with attention to the effects that can be propagated over long distances and are often associated to important biological functions. In this context, approaches inspired by network analysis can make an important contribution to the analysis of molecular dynamics simulations