Structure and kinetic properties of human D-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.
Molla G, Chaves-Sanjuan A, et al. The FASEB J. 2020 [PubMed: 31914658]
The structural bases for agonist diversity in an Arabidopsis thaliana glutamate receptor-like channel.
Alfieri A et al., Proc Natl Acad Sci USA. 2020 [PubMed: 31871183]
Structure of a bacterial ice binding protein with two faces of interaction with ice.
Mangiagalli et al., FEBS J. 2018 [Pubmed: 29533528]
Fusicoccin activates KAT1 channels by stabilizing their interaction with 14-3-3 proteins.
Saponaro et al., Plant Cell. 2017 [PubMed: 28970335]
Crystal structure of the Arabidopsis thaliana L1L/NF-YC3 histone-fold dimer reveals specificities of the LEC1 family of NF-Y subunits in plants.
Gnesutta et al., Mol Plant. 2017 [PubMed: 27871811]
A redox signaling globin is essential for reproduction in Caenorhabditis elegans.
De Henau et al., Nature Commun. 2015 [PubMed: 26621324]
Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.
Lolicato et al., Nature Chem Biol. 2014 [PubMed: 24776929]
Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination.
Nardini et al., Cell. 2013 [PubMed: 23332751]
Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS.
Colanzi et al., Proc Natl Acad Sci USA. 2013 [PubMed: 23716697]
Archaeal protoglobin structure indicates new ligand diffusion paths and modulation of haem-reactivity.
Nardini et al., EMBO Rep. 2008 [PubMed: 18188182]
Cold adapted proteins
The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs).
Chaves-Sanjuan A, Gnesutta N, Gobbini A, Martignago D, Bernardini A, Fornara F, Mantovani R, Nardini M. Structural determinants for NF-Y subunit organization and NF-Y/DNA association in plants. Plant J. 2020 Oct 24. doi: 10.1111/tpj.15038. Epub ahead of print. [PubMed: 33098724]
Nardone V, Chaves-Sanjuan A, Lapi M, Airoldi C, Saponaro A, Pasqualato S, Dolfini D, Camilloni C, Bernardini A, Gnesutta N, Mantovani R, Nardini M. Structural Basis of Inhibition of the Pioneer Transcription Factor NF-Y by Suramin. Cells. 2020 Oct 29;9(11):2370. doi: 10.3390/cells9112370. [PubMed: 33138093]
Bernardini A, Lorenzo M, Nardini M, Mantovani R, Gnesutta N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer. FASEB J. 2019 Apr;33(4):4790-4801. doi: 10.1096/fj.201801989R. [PubMed: 30589568]
Gnesutta N, Saad D, Chaves-Sanjuan A, Mantovani R, Nardini M. Crystal Structure of the Arabidopsis thaliana L1L/NF-YC3 Histone-fold Dimer Reveals Specificities of the LEC1 Family of NF-Y Subunits in Plants. Mol Plant. 2017 Apr 3;10(4), 645-648. DOI: 10.1016/j.molp.2016.11.006 [PubMed: 27871811]
Nardone V, Chaves-Sanjuan A, Nardini M. Structural determinants for NF-Y/DNA interaction at the CCAAT box. Biochim Biophys Acta. 2017 May;1860(5):571-580. doi: 10.1016/j.bbagrm.2016.09.006 [PubMed: 27677949]
Gnesutta N, Nardini M, Mantovani R. The H2A/H2B-like histone-fold domain proteins at the crossroad between chromatin and different DNA metabolisms. Transcription. 2013 May-Jun;4(3):114-9. [PubMed: 23756340]
Nardini M, Gnesutta N, Donati G, Gatta R, Forni C, Fossati A, Vonrhein C, Moras D, Romier C, Bolognesi M, Mantovani R. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination. Cell. 2013 Jan 17;152(1-2):132-43. doi: 10.1016/j.cell.2012.11.047. [PubMed: 23332751]
Antonio Chaves-Sanjuan, Louise Gourlay, Marco Nardini
Prof. R. Mantovani and Dr. N. Gnesutta, Dept. of Biosciences, University of Milano (Italy)
In 2013 we first reported the crystal structure of the binary complex composed of the NF-Y trimer and a CCAAT box containing a 25 bp DNA fragment (PDB-code: 4AWL). Our results detail the structural basis of a sequence specific, histone-like, mode of DNA binding by NF-Y (Nardini et al., 2013). The NF-YA subunit both binds to NF-YB/NF-YC and inserts an alpha helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways (Gnesutta et al., 2013; Nardone et al., 2017).
In plants a genetic diversification has occurred, resulting in the presence of several genes coding for each NF-Y subunit. As a consequence, many NF-Y trimeric complexes may form providing a flexible combinatorial system able to fulfill different roles in specific pathways. So far, we described the structural characterization of the Arabidopsis thaliana NF-YA6/NF-YB2/NF-YC3 in complex with its target DNA, FT CCAAT (PDB-code 6R2V), and of the histone dimers NF-YB6(L1L)/NF-YC3 (PDB-code: 5G49), NF-YB2/NF-YC3 (PDB-codes: 6R0M and 6R0N), and rice Ghd8/OsNF-YC7 (PDB-code: 6R0L) (Chaves-Sanjuan et al., 2020; Gnesutta et al., 2017).
Recently, NF-Y has been implicated in several types of cancer. Indeed, tumor cells undergo 'metabolic rewiring' through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. Within this project, we have identified suramin as a promising NF-Y inhibitor (Nardone et al., 2020).
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are K+ channels dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain.
We analysed the structural features and the dynamics associated to the isoform-specific cAMP gating in HCN channels (PDB-codes: 3U0Z, 3U10, 3U11; Lolicato et al., 2011) and we demonstrated at the atomic level how cyclic dinucleotides, an emerging class of second messengers in mammals, control channel cAMP responsiveness (PDB-code: 4KL1; Lolicato et al., 2014), and how the drug ivabradine (a specific heart rate-reducing agent) is able to block the channel (Bucchi et al., 2013).
Within the same line of research on regulation of K+ channels, very recently we showed by X-ray crystallography that the K+ inward rectifier KAT1 (K+ Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase (PDB-codes: 5NWI, 5NWJ, 5NWK). These data are of significance since they advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells (Saponaro et al., 2017).
Antonio Chaves-Sanjuan, Marco Nardini
Prof. A. Moroni, Prof. D. DiFrancesco, and Prof. M. Baruscotti, Dept. of Biosciences, University of Milano (Italy)
Saponaro A, Porro A, Chaves-Sanjuan A, Nardini M, Rauh O, Thiel G, Moroni A. Fusicoccin Activates KAT1 Channels by Stabilizing Their Interaction with 14-3-3 Proteins. Plant Cell. 2017 Oct;29(10):2570-2580. doi: 10.1105/tpc.17.00375. [PubMed: 28970335]
Lolicato M, Bucchi A, Arrigoni C, Zucca S, Nardini M, Schroeder I, Simmons K, Aquila M, DiFrancesco D, Bolognesi M, Schwede F, Kashin D, Fishwick CW, Johnson AP, Thiel G, Moroni A. Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness. Nat Chem Biol. 2014 Jun;10(6):457-62. doi: 10.1038/nchembio.1521. [PubMed: 24776929]
Bucchi A, Baruscotti M, Nardini M, Barbuti A, Micheloni S, Bolognesi M, DiFrancesco D. Identification of the molecular site of ivabradine binding to HCN4 channels. PLoS One. 2013;8(1):e53132. doi: 10.1371/journal.pone.0053132. [PubMed: 23308150]
Lolicato M, Nardini M, Gazzarrini S, Moller S, Bertinetti D, Herberg FW, Bolognesi M, Martin H, Fasolini M, Bertrand JA, Arrigoni C, Thiel G, Moroni A. Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels. J Biol Chem. 2011 Dec 30;286(52):44811-20. doi: 10.1074/jbc.M111.297606. [PubMed: 22006928]
Muscular dystrophies (MDs) are still incurable diseases, characterized by muscle wasting, replacement of fibrotic tissue, increasing weakness, which in severe cases leads to premature death. A common consensus in the field agrees that any successful therapeutic strategy has to rely on good muscle quality. Recent data indicate that making a dystrophic muscle slower in regeneration and in metabolic contraction, by silencing the transcription factor Nfix, preserves muscle, both morphologically and functionally. These results represented a research breakthrough and provided new hope for MD treatment. We are currently working on the 3D structure determination of Nfix with the aim of using it as a target for the structure-based drug discovery/design of inhibitors able to interfere with its DNA-binding properties.
Antonio Chaves-Sanjuan, Michela Lapi, Louise Gourlay, Marco Nardini
Prof. G. Messina, Dept. of Biosciences, University of Milano (Italy)
Cold adapted proteins
The work on cold-adapted proteins started a few years ago and it can be seen as a continuation of our structural work on alfa/beta-hydrolase fold enzymes and hemoproteins. So far we solved the structure of a truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PDB-code: 4UUR; Giordano et al., 2015), a cytoglobin from the Antarctic fish Dissostichus mawsoni (PDB-code: 6Q6P; Giordano et al., 2020), a beta galactosidase from the psychrophilic Marinomonas ef1 (PDB-code: 6Y2K; Mangiagalli et al., 2020), and a bacterial acyl aminoacyl peptidase from Sporosarcina psychrophila(PDB-code: 5L8S; Brocca et al., 2016). Our data shed further light on how these cold-adapted proteins developed a molecular strategy to enhance their flexibility while still preserving sufficient stability to be functional. Within this project line, we also solved the structure (at the atomic resolution of 0.84 A) of an antifreeze protein from the Antarctic ciliate Euplotes focardii and the associated bacterial consortium (PDB-code: 6EIO). These kind of proteins are able to protect from freezing damage the organisms exposed to permanent sub-zero temperatures or seasonal temperature dropping and because of these properties it has been envisaged their potential in food processing, cryopreservation, cryosurgery, fishery and agricultural industries, and anti-ice materials development. Furthermore, this protein has been used as a target for the international project of Critical Assessment of protein Structure Prediction (CASP12) (http://predictioncenter.org/) to test sequence-based 3D structural predictions of proteins (Kryshtafovych et al., 2017).
Mangiagalli M, Lapi M, Maione S, Orlando M, Brocca S, Pesce A, Barbiroli A, Camilloni C, Pucciarelli S, Lotti M, Nardini M. The co-existence of cold activity and thermal stability in an Antarctic GH42 beta-galactosidase relies on its hexameric quaternary arrangement. FEBS J. 2020 May 4. doi: 10.1111/febs.15354. Epub ahead of print. [PubMec: 32363751].
Giordano D, Pesce A, Vermeylen S, Abbruzzetti S, Nardini M, Marchesani F, Berghmans H, Seira C, Bruno S, Javier Luque F, di Prisco G, Ascenzi P, Dewilde S, Bolognesi M, Viappiani C, Verde C. Structural and functional properties of Antarctic fish cytoglobins-1: Cold-reactivity in multi-ligand reactions. Comput Struct Biotechnol J. 2020 Aug 12;18:2132-2144. doi: 10.1016/j.csbj.2020.08.007. [PMID: 32913582]
Kryshtafovych A, Albrecht R, Basle A, Bule P, Caputo AT, Carvalho AL, Chao KL, Diskin R, Fidelis K, Fontes CMGA, Fredslund F, Gilbert HJ, Goulding CW, Hartmann MD, Hayes CS, Herzberg O, Hill JC, Joachimiak A, Kohring GW, Koning RI, Lo Leggio L, Mangiagalli M, Michalska K, Moult J, Najmudin S, Nardini M, Nardone V, Ndeh D, Nguyen TH, Pintacuda G, Postel S, van Raaij MJ, Roversi P, Shimon A, Singh AK, Sundberg EJ, Tars K, Zitzmann N, Schwede T. Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016). Proteins. 2017 Sep 28. doi: 10.1002/prot.25392. [Epub ahead of print] [PubMed: 28960539]
Brocca S, Ferrari C, Barbiroli A, Pesce A, Lotti M, Nardini M. A bacterial acyl aminoacyl peptidase couples flexibility and stability as a result of cold adaptation. FEBS J. 2016 Dec;283(23):4310-4324. doi: 10.1111/febs.13925. [PubMed: 27739253]
Giordano D, Pesce A, Boechi L, Bustamante JP, Caldelli E, Howes BD, Riccio A, di Prisco G, Nardini M, Estrin D, Smulevich G, Bolognesi M, Verde C. Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. FEBS J. 2015 Aug;282(15):2948-65. doi: 10.1111/febs.13335. [PubMed: 26040838]
Prof. A. Pesce, University of Genova (Italy); Dr. C. Verde, CNR-IBBR Naples (Italy); Prof. M. Lotti, University of Milano-Bicocca (Italy)
Nardini's lab is involved in the study of flavoproteins and other enzymes and their involvement in several human pathologies. In addition, several wild-type and variants of enzymes have been studied for their potential in biotechnological applications. The two main lines of research are focused on FAD-containing enzymes involved in the L-amino acid and D-amino acid oxidation.
Regarding L-amino acid oxidases, so far we solved the structure of L-Amino acid deaminase from Proteus myxofaciens (PmaLAAD), and aminoacetone oxidase from Streptococcus oligofermentans (SoAAO). PmaLAAD is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic L-amino acids into keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Our structural studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids (PDB-codes: 5FJM and 5FJN; Motta et al., 2016). SoAAO represents, instead, a member of a novel family of bacterial flavoproteins involved in specific functions, such as participating in antioxidant defence from the pro-oxidant metabolite aminoacetone
Our results show that the SoAAO fold shows a new three-domain architecture, unrelated to that of LAAOs, and it doesn't display LAAO activity. Such results support a specialized role for SoAAO in the microbial defence mechanism related to aminoacetone catabolism through a mechanism yielding dimethylpyrazine derivatives instead of methylglyoxal (PDB-codes: 4CNJ and 4CNK; Molla et al., 2014).
Molla G, Chaves-Sanjuan A, Savinelli A, Nardini M, Pollegioni L. Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain. FASEB J. 2020 Jan;34(1):1182-1197. doi: 10.1096/fj.201901703R. PMID: 31914658.
Motta P, Molla G, Pollegioni L, Nardini M. Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes. J Biol Chem. 2016 May 13;291(20):10457-75. doi: 10.1074/jbc.M115.703819. PMID: 27022028
Molla G, Nardini M, Motta P, D'Arrigo P, Panzeri W, Pollegioni L. Aminoacetone oxidase from Streptococcus oligofermentans belongs to a new three-domain family of bacterial flavoproteins. Biochem J. 2014 Dec 15;464(3):387-99. doi: 10.1042/BJ20140972. PMID: 25269103.
Hopkins SC, Heffernan ML, Saraswat LD, Bowen CA, Melnick L, Hardy LW, Orsini MA, Allen MS, Koch P, Spear KL, Foglesong RJ, Soukri M, Chytil M, Fang QK, Jones SW, Varney MA, Panatier A, Oliet SH, Pollegioni L, Piubelli L, Molla G, Nardini M, Large TH. Structural, kinetic, and pharmacodynamic mechanisms of D-amino acid oxidase inhibition by small molecules. J Med Chem. 2013 May 9;56(9):3710-24. doi: 10.1021/jm4002583. PMID: 23631755.
Marco Nardini, Antonio Chaves-Sanjuan
Prof. L. Pollegioni and Prof. G. Molla, University of Insubria (Italy)
On the D-amino acid oxidase side, we characterized the mechanism and pharmacodynamics of five structurally distinct inhibitors of human D-amino acid oxidase (hDAAO). DAAO activity plays an important role in the regulation of NMDAR neurotransmission. In particular, DAAO activity may physiologically limit NMDAR activity by decreasing the local concentrations of D-serine within the astrocytic environment of neurons and synapses in defined brain regions. The therapeutic potential for small molecule inhibitors to treat disorders like schizophrenia or neuropathic pain requires a better understanding of pharmacodynamics and mechanism of DAAO inhibition and of its physiological role in NMDAR function (PDB-codes: 3ZNN, 3ZNO, 3ZNQ, and 3ZNP; Hopkins et al., 2013).
The latest entry on our structural gallery is human D-Asp oxidase (hDASPO). The enzyme catalyzes the oxidative deamination of D-Asp
to yield oxaloacetate, ammonia, and hydrogen peroxide. D-Asp features many of the signatures of a classical neurotransmitter, acting as an agonist on postsynaptic NMDAR. Our results highlight peculiar features of hDASPO relevant for controlling d-Asp cellular levels and have now paved
the way for the challenge to rationally design inhibitors that could reduce d-Asp catabolism and potentially serve as novel