BMC Structural Biology
The latest research articles published by BMC Structural Biology
An intact helical domain is required for Gα <sub>14</sub> to stimulate phospholipase Cβ
Background: Stimulation of phospholipase Cβ (PLCβ) by the activated α-subunit of G q (Gα q ) constitutes a major signaling pathway for cellular regulation, and structural studies have recently revealed the molecular interactions between PLCβ and Gα q . Yet, most of the PLCβ-interacting residues identified on Gα q are not unique to members of the Gα q family. Molecular modeling predicts that the core PLCβ-interacting residues located on the switch regions of Gα q are similarly positioned in Gα z which does not stimulate PLCβ. Using wild-type and constitutively active chimeras constructed between Gα z and Gα 14 , a member of the Gα q family, we examined if the PLCβ-interacting residues identified in Gα q are indeed essential. Results: Four chimeras with the core PLCβ-interacting residues composed of Gα z sequences were capable of binding PLCβ2 and stimulating the formation of inositol trisphosphate. Surprisingly, all chimeras with a Gα z N-terminal half failed to functionally associate with PLCβ2, despite the fact that many of them contained the core PLCβ-interacting residues from Gα 14 . Further analyses revealed that the non-PLCβ2 interacting chimeras were capable of interacting with other effector molecules such as adenylyl cyclase and tetratricopeptide repeat 1, indicating that they could adopt a GTP-bound active conformation. Conclusion: Collectively, our study suggests that the previously identified PLCβ-interacting residues are insufficient to ensure productive interaction of Gα 14 with PLCβ, while an intact N-terminal half of Gα 14 is apparently required for PLCβ interaction.
The crystal structure of JNK from <it>Drosophila melanogaster</it> reveals an evolutionarily conserved topology with that of mammalian JNK proteins
Background: The c-Jun N-terminal kinases (JNKs), members of the mitogen-activated protein kinase (MAPK) family, engage in diverse cellular responses to signals produced under normal development and stress conditions. In Drosophila, only one JNK member is present, whereas ten isoforms from three JNK genes (JNK1, 2, and 3) are present in mammalian cells. To date, several mammalian JNK structures have been determined, however, there has been no report of any insect JNK structure. Results: We report the first structure of JNK from Drosophila melanogaster (DJNK). The crystal structure of the unphosphorylated form of DJNK complexed with adenylyl imidodiphosphate (AMP-PNP) has been solved at 1.79 Å resolution. The fold and topology of DJNK are similar to those of mammalian JNK isoforms, demonstrating their evolutionarily conserved structures and functions. Structural comparisons of DJNK and the closely related mammalian JNKs also allow identification of putative catalytic residues, substrate-binding sites and conformational alterations upon docking interaction with Drosophila scaffold proteins. Conclusions: The DJNK structure reveals common features with those of the mammalian JNK isoforms, thereby allowing the mapping of putative catalytic and substrate binding sites. Additionally, structural changes upon peptide binding could be predicted based on the comparison with the closely-related JNK3 structure in complex with pepJIP1. This is the first structure of insect JNK reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of insect JNK.
Free fatty acid receptors: structural models and elucidation of ligand binding interactions
Background: The free fatty acid receptors (FFAs), including FFA1 (orphan name: GPR40), FFA2 (GPR43) and FFA3 (GPR41) are G protein-coupled receptors (GPCRs) involved in energy and metabolic homeostasis. Understanding the structural basis of ligand binding at FFAs is an essential step toward designing potent and selective small molecule modulators. Results: We analyse earlier homology models of FFAs in light of the newly published FFA1 crystal structure co-crystallized with TAK-875, an ago-allosteric ligand, focusing on the architecture of the extracellular binding cavity and agonist-receptor interactions. The previous low-resolution homology models of FFAs were helpful in highlighting the location of the ligand binding site and the key residues for ligand anchoring. However, homology models were not accurate in establishing the nature of all ligand-receptor contacts and the precise ligand-binding mode. From analysis of structural models and mutagenesis, it appears that the position of helices 3, 4 and 5 is crucial in ligand docking. The FFA1-based homology models of FFA2 and FFA3 were constructed and used to compare the FFA subtypes. From docking studies we propose an alternative binding mode for orthosteric agonists at FFA1 and FFA2, involving the interhelical space between helices 4 and 5. This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42. The novel FFAs structural models highlight higher aromaticity of the FFA2 binding cavity and higher hydrophilicity of the FFA3 binding cavity. The role of the residues at the second extracellular loop used in mutagenesis is reanalysed. The third positively-charged residue in the binding cavity of FFAs, located in helix 2, is identified and predicted to coordinate allosteric modulators. Conclusions: The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions. Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.
Structural characterization of the carbohydrate-binding module of NanA sialidase, a pneumococcal virulence factor
Background: Streptococcus pneumoniae Neuraminidase A (NanA) is a multi-domain protein anchored to the bacterial surface. Upstream of the catalytic domain of NanA is a domain that conforms to the sialic acid-recognising CBM40 family of the CAZY (carbohydrate-active enzymes) database. This domain has been identified to play a critical role in allowing the bacterium to promote adhesion and invasion of human brain microvascular endothelial cells, and hence may play a key role in promoting bacterial meningitis. In addition, the CBM40 domain has also been reported to activate host chemokines and neutrophil recruitment during infection. Results: Crystal structures of both apo- and holo- forms of the NanA CBM40 domain (residues 121 to 305), have been determined to 1.8 Å resolution. The domain shares the fold of other CBM40 domains that are associated with sialidases. When in complex with α2,3- or α2,6-sialyllactose, the domain is shown to interact only with the terminal sialic acid. Significantly, a deep acidic pocket adjacent to the sialic acid-binding site is identified, which is occupied by a lysine from a symmetry-related molecule in the crystal. This pocket is adjacent to a region that is predicted to be involved in protein-protein interactions. Conclusions: The structural data provide the details of linkage-independent sialyllactose binding by NanA CBM40 and reveal striking surface features that may hold the key to recognition of binding partners on the host cell surface. The structure also suggests that small molecules or sialic acid analogues could be developed to fill the acidic pocket and hence provide a new therapeutic avenue against meningitis caused by S. pneumoniae.
Background: Sec4p is a small monomeric Ras-related GTP-binding protein (23 kDa) that regulates polarized exocytosis in S. cerevisiae. In this study we examine the structural effects of a conserved serine residue in the P-loop corresponding to G12 in Ras. Results: We show that the Sec4p residue serine 29 forms a hydrogen bond with the nucleotide. Mutations of this residue have a different impact than equivalent mutations in Ras and can form stable associations with the exchange factor allowing us to elucidate the structure of a complex of Sec4p bound to the exchange factor Sec2p representing an early stage of the exchange reaction. Conclusions: Our structural investigation of the Sec4p-Sec2p complex reveals the role of the Sec2p coiled-coil domain in facilitating the fast kinetics of the exchange reaction. For Ras-family GTPases, single point mutations that impact the signaling state of the molecule have been well described however less structural information is available for equivalent mutations in the case of Rab proteins. Understanding the structural properties of mutants such as the one described here, provides useful insights into unique aspects of Rab GTPase function.
Crystal structure of <it>O</it>-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution
Background: Calicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL. Results: Crystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product). Conclusions: Likely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.
Background: Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain. Results: The first structure of a Firmicute Sps protein YuiC from B. subtilis, is found to be a stripped down version of the cell-wall peptidoglycan hydrolase MltA. The YuiC structures are of a domain swapped dimer, although some monomer is also found in solution. The protein crystallised in the presence of pentasaccharide shows a 1,6-anhydrodisaccharide sugar product, indicating that YuiC cleaves the sugar backbone to form an anhydro product at least on lengthy incubation during crystallisation. Conclusions: The structural simplification of MltA in Sps proteins is analogous to that of the resuscitation promoting factor domains of Actinobacteria, which are stripped down versions of lysozyme and soluble lytic transglycosylase proteins.
Variations in periplasmic loop interactions determine the pH-dependent activity of the hexameric urea transporter UreI from Helicobacter pylori: a molecular dynamics study
Background: Helicobacter pylori is an important factor in the development of diseases such as ulcer and gastric cancer. This bacterium uses a periplasmic transporter, UreI, to deliver urea to the intracelullar space, where later it is transformed into ammonia by the cytoplasmic enzyme urease to survive the acidic condition of the human stomach. The UreI transporter presents a pH-dependent activity, where this pH-dependence remains unknown at a structural level. Althought the existance of several protonable residues in the periplasmic loops are related to the pH-dependent activity, we find interesting to have a clear view of the conformational changes involved in this phenomena through a molecular dynamic study. Results: Molecular dynamic simulations of the UreI transporter at three different pH conditions were performed, revealing two main pH-dependent conformations, which we present as the open and close states. We find that salt bridges between the periplasmic loops are crucial interactions that stabilize these conformations. Besides, a cooperative behaviour exists between the six subunits of the system that is necessary to fulfill the activity of this transporter. Conclusions: We found different pH-dependent conformations of the urea transporter UreI from Helicobacter pylori, which are related to salt-bridge interactions in the periplasmic regions. The behaviour of every channel in the system is not independent, given the existance of a cooperative behaviour through the formation of salt-bridges between the subunits of the hexameric system. We believe that our results will be related to the generation of new eradication therapies using this transporter as an attractive target, denoting that the knowledge of the possible pH-dependent conformations adopted for this transporter are important for the development of rational drug design approximations.