Institute of Biostructures and Bioimaging

Napoli, Italy

Institute of Biostructures and Bioimaging

Napoli, Italy
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Nikitushkin V.D.,RAS A.N. Bach Institute of Biochemistry | Demina G.R.,RAS A.N. Bach Institute of Biochemistry | Shleeva M.O.,RAS A.N. Bach Institute of Biochemistry | Guryanova S.V.,RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry | And 3 more authors.
FEBS Journal | Year: 2015

Resuscitation-promoting factor proteins (Rpfs) are known to participate in reactivating the dormant forms of actinobacteria. Structural analysis of the Rpf catalytic domain demonstrates its similarity to lysozyme and to lytic transglycosylases - the groups of enzymes that cleave the β-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc, and concomitantly form a 1,6-anhydro ring at the MurNAc residue. Analysis of the products formed from mycobacterial peptidoglycan hydrolysis reactions containing a mixture of RpfB and resuscitation-promoting factor interacting protein (RipA) allowed us to identify the suggested product of their action - N-acetylglucosaminyl-β(1→4)-N-glycolyl-1,6-anhydromuramyl-l-alanyl-d-isoglutamate. To identify the role of this resulting product in resuscitation, we used a synthetic 1,6-anhydrodisaccharide-dipeptide, and tested its ability to stimulate resuscitation by using the dormant Mycobacterium smegmatis model. It was found that the disaccharide-dipeptide was the minimal structure capable of resuscitating the dormant mycobacterial cells over the concentration range of 9-100 ng·mL-1. The current study therefore provides the first insights into the molecular mechanism of resuscitation from dormancy involving a product of RpfB/RipA-mediated peptidoglycan cleavage. © 2015 FEBS.


PubMed | RAS A.N. Bach Institute of Biochemistry, Institute of Biostructures and Bioimaging and RAS Shemyakin Ovchinnikov Institute of Bioorganic Chemistry
Type: Journal Article | Journal: The FEBS journal | Year: 2015

Resuscitation-promoting factor proteins (Rpfs) are known to participate in reactivating the dormant forms of actinobacteria. Structural analysis of the Rpf catalytic domain demonstrates its similarity to lysozyme and to lytic transglycosylases - the groups of enzymes that cleave the -1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc, and concomitantly form a 1,6-anhydro ring at the MurNAc residue. Analysis of the products formed from mycobacterial peptidoglycan hydrolysis reactions containing a mixture of RpfB and resuscitation-promoting factor interacting protein (RipA) allowed us to identify the suggested product of their action - N-acetylglucosaminyl-(1 4)-N-glycolyl-1,6-anhydromuramyl-L-alanyl-D-isoglutamate. To identify the role of this resulting product in resuscitation, we used a synthetic 1,6-anhydrodisaccharide-dipeptide, and tested its ability to stimulate resuscitation by using the dormant Mycobacterium smegmatis model. It was found that the disaccharide-dipeptide was the minimal structure capable of resuscitating the dormant mycobacterial cells over the concentration range of 9-100 ng mL(-1). The current study therefore provides the first insights into the molecular mechanism of resuscitation from dormancy involving a product of RpfB/RipA-mediated peptidoglycan cleavage.


Esposito C.,Institute of Biostructures and Bioimaging | Carullo P.,University of Naples Federico II | Pedone E.,Institute of Biostructures and Bioimaging | Graziano G.,University of Sannio | And 2 more authors.
FEBS Letters | Year: 2010

Heparin Binding Hemagglutinin A (HBHA) is hitherto the sole virulence factor associated with tuberculosis dissemination from the lungs, the site of primary infection, to epithelial cells. We have previously reported the solution structure of HBHA, a dimeric and elongated molecule. Since oligomerisation of HBHA is associated with its ability to induce bacterial agglutination, we investigated this process using experimental and modelling techniques. We here identified a short segment of HBHA whose presence is mandatory for the stability of folded conformation, whose denaturation is a reversible two-state process. Our data suggest that agglutination-driven cell-cell interactions do not occur via association of HBHA monomers, nor via association of HBHA dimers and open the scenario to a possible trans-dimerisation process. © 2010 Federation of European Biochemical Societies.


PubMed | Institute of Biostructures and Bioimaging, IRCCS SDN and University of Hamburg
Type: Letter | Journal: FEBS letters | Year: 2016

Potassium channel tetramerization domain-containing (KCTD) proteins are involved in fundamental physio-pathological processes. Here, we report an analysis of the oligomeric state of the Bric--brack, Tram-track, Broad complex (BTB) domains of seven distinct KCTDs belonging to five major clades of the family evolution tree. Despite their functional and sequence variability, present electron microscopy data highlight the occurrence of well-defined pentameric states for all domains. Our data also show that these states coexist with alternative forms which include open pentamers. Thermal denaturation analyses conducted using KCTD1 as a model suggest that, in these proteins, different domains cooperate to their overall stability. Finally, negative-stain electron micrographs of KCTD6(BTB) in complex with Cullin3 show the presence of assemblies with a five-pointed pinwheel shape.


PubMed | Institute of Biostructures and Bioimaging, IRCCS SDN and German Electron Synchrotron
Type: | Journal: Biochimie | Year: 2016

The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, invitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest thatINC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.


Balasco N.,Institute of Biostructures and Bioimaging | Balasco N.,The Second University of Naples | Barone D.,Institute of Biostructures and Bioimaging | Barone D.,The Second University of Naples | Vitagliano L.,Institute of Biostructures and Bioimaging
Journal of Biomolecular Structure and Dynamics | Year: 2015

Recent structural investigations have shown that the C-terminal domain (CTD) of the transcription factor RfaH undergoes unique structural modifications that have a profound impact into its functional properties. These modifications cause a complete change in RfaHCTD topology that converts from an α-hairpin to a β-barrel fold. To gain insights into the determinants of this major structural conversion, we here performed computational studies (protein structure prediction and molecular dynamics simulations) on RfaHCTD. Although these analyses, in line with literature data, suggest that the isolated RfaHCTD has a strong preference for the β-barrel fold, they also highlight that a specific region of the protein is endowed with a chameleon conformational behavior. In particular, the Leu-rich region (residues 141-145) has a good propensity to adopt both α-helical and β-structured states. Intriguingly, in the RfaH homolog NusG, whose CTD uniquely adopts the β-barrel fold, the corresponding region is rich in residues as Val or Ile that present a strong preference for the β-structure. On this basis, we suggest that the presence of this Leu-rich element in RfaHCTD may be responsible for the peculiar structural behavior of the domain. The analysis of the sequences of RfaH family (PfamA code PF02357) unraveled that other members potentially share the structural properties of RfaHCTD. These observations suggest that the unusual conformational behavior of RfaHCTD may be rare but not unique. © 2015 © 2015 Taylor and Francis.


Smaldone G.,IRCCS SDN Naples Italy | Pirone L.,Institute of Biostructures and Bioimaging | Pedone E.,Institute of Biostructures and Bioimaging | Marlovits T.,German Electron Synchrotron | And 2 more authors.
FEBS Letters | Year: 2016

Potassium channel tetramerization domain-containing (KCTD) proteins are involved in fundamental physio-pathological processes. Here, we report an analysis of the oligomeric state of the Bric-à-brack, Tram-track, Broad complex (BTB) domains of seven distinct KCTDs belonging to five major clades of the family evolution tree. Despite their functional and sequence variability, present electron microscopy data highlight the occurrence of well-defined pentameric states for all domains. Our data also show that these states coexist with alternative forms which include open pentamers. Thermal denaturation analyses conducted using KCTD1 as a model suggest that, in these proteins, different domains cooperate to their overall stability. Finally, negative-stain electron micrographs of KCTD6BTB in complex with Cullin3 show the presence of assemblies with a five-pointed pinwheel shape. © 2016 Federation of European Biochemical Societies.


Squeglia F.,Institute of Biostructures and Bioimaging | Squeglia F.,University of Naples Federico II | Romano M.,Institute of Biostructures and Bioimaging | Romano M.,The Second University of Naples | And 4 more authors.
Biophysical Journal | Year: 2013

Resuscitation of Mtb is crucial to the etiology of Tuberculosis, because latent tuberculosis is estimated to affect one-third of the world population. The resuscitation-promoting factor RpfB is mainly responsible for Mtb resuscitation from dormancy. Given the impact of latent Tuberculosis, RpfB represents an interesting target for tuberculosis drug discovery. However, no molecular models of substrate binding and catalysis are hitherto available for this enzyme. Here, we identified key interactions involved in substrate binding to RpfB by combining x-ray diffraction studies and computational approaches. The crystal structure of RpfB catalytic domain in complex with N,N′,N″- triacetyl-chitotriose, as described here, provides the first, to our knowledge, atomic representation of ligand recognition by RpfB and demonstrates that the strongest interactions are established by the N-acetylglucosamine moiety in the central region of the enzyme binding cleft. Molecular dynamics analyses provided information on the dynamic behavior of protein-substrate interactions and on the role played by the solvent in RpfB function. These data combined with sequence conservation analysis suggest that Glu-292 is the sole residue crucial for catalysis, implying that RpfB acts via the formation of an oxocarbenium ion rather than a covalent intermediate. Present data represent a solid base for the design of effective drug inhibitors of RpfB. Moreover, homology models were generated for the catalytic domains of all members of the Mtb Rpf family (RpfA-E). The analysis of these models unveiled analogies and differences among the different members of the Rpf protein family. © 2013 Biophysical Society.


Balasco N.,Institute of Biostructures and Bioimaging | Balasco N.,The Second University of Naples | Pirone L.,Institute of Crystallography | Smaldone G.,Institute of Biostructures and Bioimaging | And 4 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2014

Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5-Cul3)5 pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity. © 2014 Elsevier B.V.


PubMed | Institute of Biostructures and Bioimaging, The Second University of Naples, University of Rome La Sapienza, Institute of Crystallography and Interuniversity Center for Research on Bioactive Peptides
Type: Journal Article | Journal: PloS one | Year: 2015

Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3-BTB interface by using the intriguing approach of stabilizing the -helical conformation of Cul3-based peptides through the stapling with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49-68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300-600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3.

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