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Dive into the research topics where Flavia Squeglia is active.

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Featured researches published by Flavia Squeglia.


Proceedings of the National Academy of Sciences of the United States of America | 2014

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

Masahiro Hayafune; Rita Berisio; Roberta Marchetti; Alba Silipo; Miyu Kayama; Yoshitake Desaki; Sakiko Arima; Flavia Squeglia; Alessia Ruggiero; Ken Tokuyasu; Antonio Molinaro; Hanae Kaku; Naoto Shibuya

Significance Chitin perception by plant receptors triggers various defense responses important for plant immunity. We show the molecular basis of chitin recognition by the rice receptor, CEBiP (chitin-elicitor binding protein), and following receptor dimerization based on the results of biochemical studies, epitope mapping by saturation transfer difference NMR spectroscopy and molecular modeling/docking studies. These results clearly indicated that two CEBiP molecules simultaneously bind to one N-acetylchitoheptaose/octaose from the opposite side, through a binding site in the central lysin motif region, resulting in the dimerization of CEBiP. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving CEBiP and Oryza sativa chitin-elicitor receptor kinase 1 (OsCERK1). Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)8-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNβ1,4GlcNAc)4, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.


Journal of the American Chemical Society | 2011

Chemical Basis of Peptidoglycan Discrimination by PrkC, a Key Kinase Involved in Bacterial Resuscitation from Dormancy

Flavia Squeglia; Roberta Marchetti; Alessia Ruggiero; Rosa Lanzetta; Daniela Marasco; Jonathan Dworkin; Maxim V. Petoukhov; Antonio Molinaro; Rita Berisio; Alba Silipo

Bacterial Ser/Thr kinases modulate a wide number of cellular processes. In Bacillus subtilis , the Ser/Thr kinase PrkC has been shown to induce germination of bacterial spores in response to DAP-type but not Lys-type cell wall muropeptides. Muropeptides are a clear molecular signal that growing conditions are promising, since they are produced during cell wall peptidoglycan remodeling associated with cell growth and division of neighboring bacteria. However, whether muropeptides are able to bind the protein physically and how the extracellular region is able to distinguish the two types of muropeptides remains unclear. Here we tackled the important question of how the extracellular region of PrkC (EC-PrkC) senses muropeptides. By coupling NMR techniques and protein mutagenesis, we exploited the structural requirements necessary for recognition and binding and proved that muropeptides physically bind to EC-PrkC through DAP-moiety-mediated interactions with an arginine residue, Arg500, belonging to the protein C-terminal PASTA domain. Notably, mutation of this arginine completely suppresses muropeptide binding. Our data provide the first molecular clues into the mechanism of sensing of muropeptides by PrkC.


Current Protein & Peptide Science | 2012

Bacterial Cell Division Regulation by Ser/Thr Kinases: A Structural Perspective

Alessia Ruggiero; Paola De Simone; Giovanni Smaldone; Flavia Squeglia; Rita Berisio

Recent genetic, biochemical and structural studies have established that eukaryotic-like Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases are widespread in gram-positive bacteria. These data underline a key role of reversible Ser/Thr phosphorylation in bacterial physiology and virulence. Numerous studies have revealed how phosphorylation/dephosphorylation of Ser/Thr protein-kinases governs cell division and cell wall biosynthesis and that Ser/Thr protein kinases are responsible for distinct phenotypes, dependent on different environmental signals. In this review we discuss the current understandings of Ser/Thr protein-kinases functional processes based on structural data.


Biopolymers | 2014

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis

Luisa Calvanese; Lucia Falcigno; Cira Maglione; Daniela Marasco; Alessia Ruggiero; Flavia Squeglia; Rita Berisio; Gabriella D'Auria

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C‐terminal PASTA domain. Since PASTA modules are typically considered as β‐lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the β‐lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or β‐lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable.


Viruses | 2016

Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy

Grażyna Majkowska-Skrobek; Agnieszka Łątka; Rita Berisio; Barbara Maciejewska; Flavia Squeglia; Maria Fiammetta Romano; Rob Lavigne; Carsten Struve; Zuzanna Drulis-Kawa

The rise of antibiotic-resistant Klebsiella pneumoniae, a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36), from the Siphoviridae family. To gain insights into the catalytic and structural features of depoKP36, we have recombinantly produced this protein of 93.4 kDa and showed that it is able to hydrolyze a crude exopolysaccharide of a K. pneumoniae host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical K. pneumoniae strains, representing the K63 type, and significantly inhibited Klebsiella-induced mortality of Galleria mellonella larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of Klebsiella strains. The activity of this enzyme was retained in a broad range of pH values (4.0–7.0) and temperatures (up to 45 °C). Consistently, the circular dichroism (CD) spectroscopy revealed a highly stability with melting transition temperature (Tm) = 65 °C. In contrast to other phage tailspike proteins, this enzyme was susceptible to sodium dodecyl sulfate (SDS) denaturation and proteolytic cleavage. The structural studies in solution showed a trimeric arrangement with a high β-sheet content. Our findings identify depoKP36 as a suitable candidate for the development of new treatments for K. pneumoniae infections.


Protein and Peptide Letters | 2012

Enhanced Crystallizability by Protein Engineering Approaches: A General Overview

Alessia Ruggiero; Giovanni Smaldone; Flavia Squeglia; Rita Berisio

The limiting step in macromolecular crystallography is the preparation protein crystals suitable for X-ray diffraction studies. A strong prerequisite for the success of crystallization experiments is the ability to produce monodisperse and properly folded protein samples. Since the production of most protein is usually achieved using recombinant methods, it has become possible to engineer target proteins with increased propensities to form well diffracting crystals. Recent advances in bioinformatics, which takes advantage from an enhanced information in the protein databases, are of enormous help for the design of modified proteins. Based on bioinformatics analyses, the reduction of the structural complexity of proteins or their site-specific mutagenesis has proven to have a dramatic impact on both the yield of heterologous protein expression and its crystallizability. Therefore, protein engineering represents a valid tool which supports the classical crystallization screenings with a more rational approach. This review describes key methods of protein-engineering and provides a number of examples of their successful use in crystallization. Scope of proposed topic: This Topic is focused on state-of-art protein engineering techniques to increase the propensity of proteins to form crystals with suitable X-ray diffraction properties. Protein engineering methods have proven to be of great help for the crystallization of difficult targets. We herein review molecular biology and chemical methods to help protein crystallization.


Journal of Biological Chemistry | 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

Flavia Squeglia; Beth A. Bachert; Alfonso De Simone; Slawomir Lukomski; Rita Berisio

Background: Streptococcal collagen-like proteins play crucial roles in host adhesion, host cell entry, and immunomodulation of host defenses. Results: This study provides the first three-dimensional structural description of a streptococcal collagen-like protein. Conclusion: The crystal structure evidences a six-helical bundle fold, which is unusual in bacterial proteins and characteristic of viral fusion proteins. Significance: The high resolution structure provides a structural basis for the design of inhibitors of streptococcal invasion. 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.


Journal of Biomolecular Structure & Dynamics | 2017

Structure and dynamics of the multi-domain resuscitation promoting factor RpfB from Mycobacterium tuberculosis

Alessia Ruggiero; Flavia Squeglia; Maria Romano; Luigi Vitagliano; Alfonso De Simone; Rita Berisio

RpfB is multidomain protein that is crucial for Mycobacterium tuberculosis resuscitation from dormancy. This protein cleaves cell wall peptidoglycan, an essential bacterial cell wall polymer formed by glycan chains of β-(1-4)-linked-N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) cross-linked by short peptide stems. RpfB is structurally complex being composed of five distinct domains, namely a catalytic, a G5 and three DUF348 domains. Here, we have undertaken a combined experimental and computation structural investigations on the entire protein to gain insights into its structure–function relationships. CD spectroscopy and light scattering experiments have provided insights into the protein fold stability and into its oligomeric state. Using the available structure information, we modeled the entire protein structure, which includes the two DUF348 domains whose structure is experimentally unknown, and we analyzed the dynamic behavior of RpfB using molecular dynamics simulations. Present results highlight an intricate mutual influence of the dynamics of the different protein domains. These data provide interesting clues on the functional role of non-catalytic domains of RpfB and on the mechanism of peptidoglycan degradation necessary to resuscitation of M. tuberculosis.


Biochimica et Biophysica Acta | 2016

The structure of Resuscitation promoting factor B from M. tuberculosis reveals unexpected ubiquitin-like domains

Alessia Ruggiero; Flavia Squeglia; Maria Romano; Luigi Vitagliano; Alfonso De Simone; Rita Berisio

BACKGROUND RpfB is a key factor in resuscitation from dormancy of Mycobacterium tuberculosis. This protein is a cell-wall glycosidase, which cleaves cell-wall peptidoglycan. RpfB is structurally complex and is composed of three types of domains, including a catalytic, a G5 and three DUF348 domains. Structural information is currently limited to a portion of the protein including only the catalytic and G5 domains. To gain insights into the structure and function of all domains we have undertaken structural investigations on a large protein fragment containing all three types of domains that constitute RpfB (RpfB3D). METHODS The structural features of RpfB3D have been investigated combining x-ray crystallography and biophysical studies. RESULTS AND CONCLUSIONS The crystal structure of RpfB3D provides the first structural characterization of a DUF348 domain and revealed an unexpected structural relationship with ubiquitin. The crystal structure also provides specific structural features of these domains explaining their frequent association with G5 domains. GENERAL SIGNIFICANCE Results provided novel insights into the mechanism of peptidoglycan degradation necessary to the resuscitation of M. tuberculosis. Features of the DUF348 domain add structural data to a large set of proteins embedding this domain. Based on its structural similarity to ubiquitin and frequent association to the G5 domain, we propose to name this domain as G5-linked-Ubiquitin-like domain, UBLG5.


Molecular Microbiology | 2017

Collagen-like proteins of pathogenic streptococci.

Slawomir Lukomski; Beth A. Bachert; Flavia Squeglia; Rita Berisio

The collagen domain, which is defined by the presence of the Gly‐X‐Y triplet repeats, is amongst the most versatile and widespread known structures found in proteins from organisms representing all three domains of life. The streptococcal collagen‐like (Scl) proteins are widely present in pathogenic streptococci, including Streptococcus pyogenes, S. agalactiae, S. pneumoniae, and S. equi. Experiments and bioinformatic analyses support the hypothesis that all Scl proteins are homotrimeric and cell wall‐anchored. These proteins contain the rod‐shaped collagenous domain proximal to cell surface, as well as a variety of outermost non‐collagenous domains that generally lack predicted functions but can be grouped into one of six clusters based on sequence similarity. The well‐characterized Scl1 proteins of S. pyogenes show a dichotomous switch in ligand binding between human tissue and blood environments. In tissue, Scl1 adhesin specifically recognizes the wound microenvironment, promotes adhesion and biofilm formation, decreases bacterial killing by neutrophil extracellular traps, and modulates S. pyogenes virulence. In blood, ligands include components of complement and coagulation‐fibrinolytic systems, as well as plasma lipoproteins. In all, the Scl proteins signify a large family of structurally related surface proteins, which contribute to the ability of streptococci to colonize and cause diseases in humans and animals.

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Rita Berisio

National Research Council

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Alessia Ruggiero

University of Naples Federico II

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Luigi Vitagliano

University of Naples Federico II

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Maria Fiammetta Romano

University of Naples Federico II

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Maria Romano

National Research Council

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Daniela Marasco

University of Naples Federico II

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Lucia Falcigno

University of Naples Federico II

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