I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diego A.
Leonardo
,
Italo A.
Cavini
,
Fernanda A.
Sala
,
Deborah C.
Mendonça
,
Higor
V. D. Rosa
,
Patricia S.
Kumagai
,
Edson
Crusca Jr
,
Napoleao F.
Valadares
,
Ivo A.
Marques
,
Jose
Brandao-Neto
,
Claudia E.
Munte
,
Hans R.
Kalbitzer
,
Nicolas
Soler
,
Isabel
Uson
,
Ingemar
André
,
Ana
P. U. Araujo
,
Humberto
D'Muniz Pereira
,
Richard C.
Garratt
Diamond Proposal Number(s):
[6397, 23570, 25296]
Abstract: Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross-bridges necessary for higher order structures and thereby septin function.
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Feb 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Marco Túlio Alves
Da Silva
,
Ivan Rosa E.
Silva
,
Livia Maria
Faim
,
Natália Karla
Bellini
,
Murilo Leão
Pereira
,
Ana Laura
Lima
,
Teresa Cristina Leandro
De Jesus
,
Fernanda Cristina
Costa
,
Tatiana Faria
Watanabe
,
Humberto
D'Muniz Pereira
,
Sandro Roberto
Valentini
,
Cleslei Fernando
Zanelli
,
Júlio Cesar
Borges
,
Marcio Vinicius Bertacine
Dias
,
Júlia Pinheiro Chagas
Da Cunha
,
Bidyottam
Mittra
,
Norma W.
Andrews
,
Otavio Henrique
Thiemann
Open Access
Abstract: Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite’s ER stress response.
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Oct 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.
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Sep 2020
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493, 5810]
Open Access
Abstract: Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Juliana Roverta
Torini
,
Adriano
De Freitas Fernandes
,
Vitor Hugo
Balasco Serrao
,
Larissa
Romanello
,
Louise
Bird
,
Joanne E.
Nettleship
,
Raymond J.
Owens
,
Jose
Brandao-neto
,
Ana Eliza
Zeraik
,
Ricardo
Demarco
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073, 14493]
Abstract: Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.
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Jul 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14496]
Abstract: Septins are GTP-binding proteins that will often spontaneously assemble into filaments. In some species, particularly budding yeast, it is well known that these are capable of associating with membranes in order to fulfill their cellular role as a component of the cytoskeleton. Different from other human septins, SEPT7 appears to be unique in that it is an essential component of all hetero-oligomeric complexes described to date. As a step towards understanding the molecular basis of filament assembly, here we present two high-resolution structures of the SEPT7 GTPase domain complexed with GDP. One of these reveals a previously unreported coordination for the magnesium ion involving four water molecules and only a tenuous connection to the protein. The higher resolution structures provide unambiguous insight into the interactions at the G-interface where a structural motif based on an antiparallel β-bridge allows for the rationalization of why some septins show nucleotide-dependent β-strand slippage and others do not.
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Apr 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Larissa
Romanello
,
Ana Eliza
Zeraik
,
Adriano
De Freitas Fernandes
,
Juliana Roberta
Torini
,
Louise E.
Bird
,
Joanne E.
Nettleship
,
Heather
Rada
,
Yamini
Reddivari
,
Ray J.
Owens
,
Vitor Hugo Balasco
Serrão
,
Ricardo
Demarco
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Diamond Proposal Number(s):
[5073]
Abstract: Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the “de novo” purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.
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Feb 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Juliana Roberta
Torini
,
Larissa
Romanello
,
Fernanda Aparecida Heleno
Batista
,
Vitor Hugo Balasco
Serrao
,
Muhamamd
Faheem
,
Ana Eliza
Zeraik
,
Louise
Bird
,
Joanne E.
Nettleship
,
Yamini
Reddivari
,
Ray
Owens
,
Ricardo
Demarco
,
Júlio César
Borges
,
Jose
Brandao-neto
,
Humberto
D'muniz Pereira
Open Access
Abstract: Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
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Sep 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6P) as a substrate to a fructose-6P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.
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Jul 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Andressa P. A.
Pinto
,
Humberto M.
Pereira
,
Ana E.
Zeraik
,
Heloisa
Ciol
,
Frederico M.
Ferreira
,
Jose
Brandao-neto
,
Ricardo
Demarco
,
Marcos V. A. S.
Navarro
,
Cristina
Risi
,
Vitold E.
Galkin
,
Richard C.
Garratt
,
Ana P. U.
Araujo
Diamond Proposal Number(s):
[5073]
Abstract: Septins are filament−forming GTP−binding proteins involved in many essential cellular events related to cytoskeletal dynamics and maintenance. Septins can self−assemble into hetero−complexes, which polymerize into highly organized, cell membrane−interacting filaments. The number of septin genes varies among organisms, and although their structure and function have been thoroughly studied in opisthokonts (including animals and fungi), no structural studies have been reported for other organisms. This makes the single septin from Chlamydomonas (CrSEPT) a particularly attractive model for investigating whether functional homopolymeric septin filaments also exist. CrSEPT was detected at the base of the flagella in Chlamydomonas, suggesting that CrSEPT is involved in the formation of a membrane diffusion barrier. Using transmission electron microscopy, we observed that recombinant CrSEPT forms long filaments with dimensions comparable to those of the canonical structure described for opisthokonts. The GTP−binding domain of CrSEPT purified as a nucleotide−free monomer that hydrolyzes GTP and readily binds its analog GTPγS. We also found that on nucleotide binding, CrSEPT forms dimers that are stabilized by an interface involving the ligand (G−interface). Across this interface, one monomer supplied a catalytic arginine to the opposing subunit, greatly accelerating the rate of GTP hydrolysis. This is the first report of an arginine finger observed in a septin and suggests that CrSEPT may act as its own GTP−activating protein (GAP). The finger is conserved in all algal septin sequences, suggesting a possible correlation between the ability to form homo−polymeric filaments and the accelerated rate of hydrolysis which it provides.
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May 2017
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