The structure and regulation of human muscle alpha-actinin

Citation:

Ribeiro Ede, A. J, Pinotsis N, Ghisleni A, Salmazo A, Konarev PV, Kostan J, Sjoblom B, Schreiner C, Polyansky AA, Gkougkoulia EA, et al. The structure and regulation of human muscle alpha-actinin. Cell. 2014;159:1447-60.

Abstract:

The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. alpha-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa alpha-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.

Notes:

Ribeiro, Euripedes de Almeida JrPinotsis, NikosGhisleni, AndreaSalmazo, AnitaKonarev, Petr VKostan, JuliusSjoblom, BjornSchreiner, ClaudiaPolyansky, Anton AGkougkoulia, Eirini AHolt, Mark RAachmann, Finn LZagrovic, BojanBordignon, EnricaPirker, Katharina FSvergun, Dmitri IGautel, MathiasDjinovic-Carugo, KristinaengI 1593/FWF_/Austrian Science Fund FWF/Austria279408/ERC_/European Research Council/InternationalCH/08/001/BHF_/British Heart Foundation/United KingdomMR/J010456/1/MRC_/Medical Research Council/United KingdomG0200496/MRC_/Medical Research Council/United KingdomMR/K015664/1/MRC_/Medical Research Council/United KingdomI 525/FWF_/Austrian Science Fund FWF/AustriaG0600251/MRC_/Medical Research Council/United KingdomResearch Support, Non-U.S. Gov'tCell. 2014 Dec 4;159(6):1447-60. doi: 10.1016/j.cell.2014.10.056. Epub 2014 Nov 26.

Contact Details

Biochemistry, Chemistry Department
National and Kapodistrian University of Athens
(+30) 210-727 4493
Zografou
Athens, ZipCode 157 84

Recent Publications

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The identification of multiple simultaneous orientations of small molecule inhibitors binding to a protein target is a common challenge. It has recently been reported that the conformational heterogeneity of ligands is widely underreported in the Protein Data Bank, which is likely to impede optimal exploitation to improve affinity of these ligands. Significantly less is even known about multiple binding orientations for fragments (<300 Da), although this information would be essential for subsequent fragment optimisation using growing, linking or merging and rational structure-based design. Here, we use recently reported fragment hits for the SARS-CoV-2 non-structural protein 1 (nsp1) N-terminal domain to propose a general procedure for unambiguously identifying binding orientations of 2-dimensional fragments containing either sulphur or chloro substituents within the wavelength range of most tunable beamlines. By measuring datasets at two energies, using a tunable beamline operating in vacuum and optimised for data collection at very low X-ray energies, we show that the anomalous signal can be used to identify multiple orientations in small fragments containing sulphur and/or chloro substituents or to verify recently reported conformations. Although in this specific case we identified the positions of sulphur and chlorine in fragments bound to their protein target, we are confident that this work can be further expanded to additional atoms or ions which often occur in fragments. Finally, our improvements in the understanding of binding orientations will also serve to improve the rational optimisation of SARS-CoV-2 nsp1 fragment hits.

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PLCgamma enzymes are autoinhibited in resting cells and form key components of intracellular signaling that are also linked to disease development. Insights into physiological and aberrant activation of PLCgamma require understanding of an active, membrane-bound form, which can hydrolyze inositol-lipid substrates. Here, we demonstrate that PLCgamma1 cannot bind membranes unless the autoinhibition is disrupted. Through extensive molecular dynamics simulations and experimental evidence, we characterize membrane binding by the catalytic core domains and reveal previously unknown sites of lipid interaction. The identified sites act in synergy, overlap with autoinhibitory interfaces, and are shown to be critical for the phospholipase activity in cells. This work provides direct evidence that PLCgamma1 is inhibited through obstruction of its membrane-binding surfaces by the regulatory region and that activation must shift PLCgamma1 to a conformation competent for membrane binding. Knowledge of the critical sites of membrane interaction extends the mechanistic framework for activation, dysregulation, and therapeutic intervention.

Nikos Pinotsis

Associate Professor, Chemistry