title: Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.
abstract: Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs.
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bernini
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Henrici De Angelis
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Morandi
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy; SienaBioGrafiX Srl, 53100 Siena, Italy.
authors.forename: Ottavia
authors.initials: O
authors.lastname: Spiga
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Santucci
authors.affiliation: Department of Biotechnology, University of Verona, 37134 Verona, Italy.
authors.forename: Michael
authors.initials: M
authors.lastname: Assfalg
authors.affiliation: Department of Biotechnology, University of Verona, 37134 Verona, Italy.
authors.forename: Henriette
authors.initials: H
authors.lastname: Molinari
authors.affiliation: Dipartimento di Medicina Sperimentale e Clinica, University of Florence, 50134 Florence, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Pillozzi
authors.affiliation: Dipartimento di Medicina Sperimentale e Clinica, University of Florence, 50134 Florence, Italy.
authors.forename: Annarosa
authors.initials: A
authors.lastname: Arcangeli
authors.affiliation: Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy; SienaBioGrafiX Srl, 53100 Siena, Italy. Electronic address: neri.niccolai@unisi.it.
authors.forename: Neri
authors.initials: N
authors.lastname: Niccolai
mesh_terms: D054377:Chemokine CXCL12 / Q000737:chemistry*; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels
chemical_list: D054377:Chemokine CXCL12; D003497:Cyclic N-Oxides; D011994:Recombinant Proteins; D013113:Spin Labels; C001803:tempol
keywords: Dynamic drug design; Fragment-based hotspot screening; Paramagnetic probe; Protein–protein interaction; Transient surface pocket