I03-Macromolecular Crystallography
|
Ida
Freda
,
Cécile
Exertier
,
Anna
Barile
,
Antonio
Chaves-Sanjuan
,
Mirella
Vivoli Vega
,
Misha N.
Isupov
,
Nicholas J.
Harmer
,
Elena
Gugole
,
Paolo
Swuec
,
Martino
Bolognesi
,
Anita
Scipioni
,
Carmelinda
Savino
,
Martino luigi
Di salvo
,
Roberto
Contestabile
,
Beatrice
Vallone
,
Angela
Tramonti
,
Linda Celeste
Montemiglio
Diamond Proposal Number(s):
[11945]
Open Access
Abstract: Specificity in protein–DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5′-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process. Moreover, the resolution of an apo-PdxR crystallographic structure provided a detailed description of the transition of the effector domain to the holo-PdxR form triggered by the binding of the PLP effector molecule. Binding analyses of mutated DNA sequences using both wild type and PdxR variants revealed a central role of electrostatic interactions and of the intrinsic asymmetric bending of the DNA in allosterically guiding the holo-PdxR–DNA recognition process, from the first encounter through the fully bound state. Our results detail the structure and dynamics of the PdxR–DNA complex, clarifying the mechanism governing the DNA-binding mode of the holo-PdxR and the regulation features of the MocR family of transcription factors.
|
Jun 2023
|
|
I04-Macromolecular Crystallography
|
Flavio
Di Pisa
,
Stefano
De Benedetti
,
Enrico Mario Alessandro
Fassi
,
Mauro
Bombaci
,
Renata
Grifantini
,
Angelo
Musicò
,
Roberto
Frigerio
,
Angela
Pontillo
,
Cinzia
Rigo
,
Sandra
Abelli
,
Romualdo
Grande
,
Nadia
Zanchetta
,
Davide
Mileto
,
Alessandro
Mancon
,
Alberto
Rizzo
,
Alessandro
Gori
,
Marina
Cretich
,
Giorgio
Colombo
,
Martino
Bolognesi
,
Louise Jane
Gourlay
Diamond Proposal Number(s):
[20221]
Open Access
Abstract: Chagas disease (CD) is a vector-borne parasitosis, caused by the protozoan parasite Trypanosoma cruzi, that affects millions of people worldwide. Although endemic in South America, CD is emerging throughout the world due to climate change and increased immigratory flux of infected people to non-endemic regions. Containing of the diffusion of CD is challenged by the asymptomatic nature of the disease in early infection stages and by the lack of a rapid and effective diagnostic test. With the aim of designing new serodiagnostic molecules to be implemented in a microarray-based diagnostic set-up for early screening of CD, herein, we report the recombinant production of the extracellular domain of a surface membrane antigen from T. cruzi (TcSMP) and confirm its ability to detect plasma antibodies from infected patients. Moreover, we describe its high-resolution (1.62 Å) crystal structure, to which in silico epitope predictions were applied in order to locate the most immunoreactive regions of TcSMP in order to guide the design of epitopes that may be used as an alternative to the full-length antigen for CD diagnosis. Two putative, linear epitopes, belonging to the same immunogenic region, were synthesized as free peptides, and their immunological properties were tested in vitro. Although both peptides were shown to adopt a structural conformation that allowed their recognition by polyclonal antibodies raised against the recombinant protein, they were not serodiagnostic for T. cruzi infections. Nevertheless, they represent good starting points for further iterative structure-based (re)design cycles.
|
Jan 2022
|
|
I24-Microfocus Macromolecular Crystallography
|
Stefano
De Benedetti
,
Flavio
Di Pisa
,
Enrico Mario Alessandro
Fassi
,
Marina
Cretich
,
Angelo
Musicò
,
Roberto
Frigerio
,
Alessandro
Mussida
,
Mauro
Bombaci
,
Renata
Grifantini
,
Giorgio
Colombo
,
Martino
Bolognesi
,
Romualdo
Grande
,
Nadia
Zanchetta
,
Maria Rita
Gismondo
,
Davide
Mileto
,
Alessandro
Mancon
,
Louise Jane
Gourlay
Diamond Proposal Number(s):
[5912]
Open Access
Abstract: The human parasitic disease Schistosomiasis is caused by the Schistosoma trematode flatworm that infects freshwaters in tropical regions of the world, particularly in Sub-Saharan Africa, South America, and the Far-East. It has also been observed as an emerging disease in Europe, due to increased immigration. In addition to improved therapeutic strategies, it is imperative to develop novel, rapid, and sensitive diagnostic tests that can detect the Schistosoma parasite, allowing timely treatment. Present diagnosis is difficult and involves microscopy-based detection of Schistosoma eggs in the feces. In this context, we present the 3.22 Å resolution crystal structure of the circulating antigen Serine protease inhibitor from S. mansoni (SmSPI), and we describe it as a potential serodiagnostic marker. Moreover, we identify three potential immunoreactive epitopes using in silico-based epitope mapping methods. Here, we confirm effective immune sera reactivity of the recombinant antigen, suggesting the further investigation of the protein and/or its predicted epitopes as serodiagnostic Schistosomiasis biomarkers.
|
Apr 2021
|
|
Krios III-Titan Krios III at Diamond
|
Diamond Proposal Number(s):
[19714]
Open Access
Abstract: In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.
|
Nov 2020
|
|
I04-Macromolecular Crystallography
|
Andrea
Alfieri
,
Fabrizio G.
Doccula
,
Riccardo
Pederzoli
,
Matteo
Grenzi
,
Maria Cristina
Bonza
,
Laura
Luoni
,
Alessia
Candeo
,
Neli
Romano Armada
,
Alberto
Barbiroli
,
Gianluca
Valentini
,
Thomas R.
Schneider
,
Andrea
Bassi
,
Martino
Bolognesi
,
Marco
Nardini
,
Alex
Costa
Diamond Proposal Number(s):
[1894]
Abstract: Arabidopsis thaliana glutamate receptor-like (GLR) channels are amino acid-gated ion channels involved in physiological processes including wound signaling, stomatal regulation, and pollen tube growth. Here, fluorescence microscopy and genetics were used to confirm the central role of GLR3.3 in the amino acid-elicited cytosolic Ca2+ increase in Arabidopsis seedling roots. To elucidate the binding properties of the receptor, we biochemically reconstituted the GLR3.3 ligand-binding domain (LBD) and analyzed its selectivity profile; our binding experiments revealed the LBD preference for l-Glu but also for sulfur-containing amino acids. Furthermore, we solved the crystal structures of the GLR3.3 LBD in complex with 4 different amino acid ligands, providing a rationale for how the LBD binding site evolved to accommodate diverse amino acids, thus laying the grounds for rational mutagenesis. Last, we inspected the structures of LBDs from nonplant species and generated homology models for other GLR isoforms. Our results establish that GLR3.3 is a receptor endowed with a unique amino acid ligand profile and provide a structural framework for engineering this and other GLR isoforms to investigate their physiology.
|
Jan 2020
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20221]
Abstract: We report the functional and structural characterization of trehalose-6-phosphate phosphatase (TPP), from the Gram-negative bacterium B. pseudomallei that causes melioidosis, a severe infectious disease endemic in Southeast Asia and Northern Australia. TPP is a key enzyme in the trehalose biosynthesis pathway, which plays an important role in bacterial stress responses. Due to the absence of this biosynthetic pathway in mammals, TPP has drawn attention as a potential drug target, to combat antibiotic resistance.
In this context, we present a detailed biochemical analysis of purified recombinant TPP, reporting its specific high catalytic activity toward the trehalose-6-phosphate substrate, and an absolute requirement for its Mg2+ cofactor. Furthermore, we present the crystal structure of TPP solved at 1.74 Å, revealing the canonical haloacid dehalogenase (HAD) superfamily fold and conserved substrate binding pocket, from which insights into the catalytic mechanism may be deduced. Our data represent a starting point for the rational design of antibacterial drugs.
|
Jan 2020
|
|
|
|
Michela
Bollati
,
Karin
Alvarez
,
Rene
Assenberg
,
Cecile
Baronti
,
Bruno
Carnard
,
Shelley
Cook
,
Bruno
Coutard
,
Etienne
Decroly
,
Xavier
De Lamballerie
,
Ernest A.
Gould
,
Gilda
Grard
,
Jonathan
Grimes
,
Rolf
Hilgenfeld
,
Anna M.
Jansson
,
Helene
Malet
,
Erika
Mancini
,
Eloise
Mastrangelo
,
Andrea
Mattevij
,
Mario
Milani
,
David I.
Stuart
,
Gregory
Moureau
,
Johan
Neyts
,
Raymond J.
Owens
,
Jingshan
Ren
,
Barbara
Selisko
,
Silvia
Speroni
,
Holger
Steuber
,
Torsten
Unge
,
Martino
Bolognesi
Open Access
Abstract: Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.
|
Nov 2009
|
|
|
|
B.
Coutard
,
A. E.
Gorbalenya
,
E. J.
Snijder
,
A. M.
Leontovich
,
A.
Poupon
,
X.
De Lamballerie
,
R.
Charrel
,
E. A.
Gould
,
S.
Gunther
,
H.
Norder
,
B.
Klempa
,
H.
Bourhy
,
J.
Rohayem
,
E.
L’hermite
,
P.
Nordlund
,
D. I.
Stuart
,
R. J.
Owens
,
J. M.
Grimes
,
P. A.
Tucker
,
M.
Bolognesi
,
A.
Mattevi
,
M.
Coll
,
T. A.
Jones
,
J.
Åqvist
,
T.
Unge
,
R.
Hilgenfeld
,
G.
Bricogne
,
J.
Neyts
,
P.
La Colla
,
G.
Puerstinger
,
J. P.
Gonzalez
,
E.
Leroy
,
C.
Cambillau
,
J. L.
Romette
,
B.
Canard
Abstract: Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules
|
Apr 2008
|
|
