Allosteric inactivation of an engineered optogenetic GTPase.

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

Targeting Apoptotic Pathway of Cancer Cells with Phytochemicals and Plant-Based Nanomaterials.

Apoptosis is the elimination of functionally non-essential, neoplastic, and infected cells via the mitochondrial pathway or death receptor pathway. The process of apoptosis is highly regulated through membrane channels and apoptogenic proteins. Apoptosis maintains cellular balance within the human body through cell cycle progression. Loss of apoptosis control prolongs cancer cell survival and allows the accumulation of mutations that can promote angiogenesis, promote cell proliferation, disrupt differentiation, and increase invasiveness during tumor progression. The apoptotic pathway has been extensively studied as a potential drug target in cancer treatment. However, the off-target activities of drugs and negative implications have been a matter of concern over the years. Phytochemicals (PCs) have been studied for their efficacy in various cancer cell lines individually and synergistically. The development of nanoparticles (NPs) through green synthesis has added a new dimension to the advancement of plant-based nanomaterials for effective cancer treatment. This review provides a detailed insight into the fundamental molecular pathways of programmed cell death and highlights the role of PCs along with the existing drugs and plant-based NPs in treating cancer by targeting its programmed cell death (PCD) network.

Mapping allosteric communications within individual proteins.

Allostery in proteins influences various biological processes such as regulation of gene transcription and activities of enzymes and cell signaling. Computational approaches for analysis of allosteric coupling provide inexpensive opportunities to predict mutations and to design small-molecule agents to control protein function and cellular activity. We develop a computationally efficient network-based method, Ohm, to identify and characterize allosteric communication networks within proteins. Unlike previously developed simulation-based approaches, Ohm relies solely on the structure of the protein of interest. We use Ohm to map allosteric networks in a dataset composed of 20 proteins experimentally identified to be allosterically regulated. Further, the Ohm allostery prediction for the protein CheY correlates well with NMR CHESCA studies. Our webserver, Ohm.dokhlab.org, automatically determines allosteric network architecture and identifies critical coupled residues within this network.

The structure of MP-4 from Mucuna pruriens at 2.22†Šresolution.

The structure of the MP-4 protein was previously determined at a resolution of 2.8 Å. Owing to the unavailability of gene-sequence information at the time, the side-chain assignment was carried out on the basis of a partial sequence available through Edman degradation, sequence homology to orthologs and electron density. The structure of MP-4 has now been determined at a higher resolution (2.22 Å) in another space group and all of the structural inferences that were presented in the previous report of the structure were validated. In addition, the present data allowed an improved assignment of side chains and enabled further analysis of the MP-4 structure, and the accuracy of the assignment was confirmed by the recently available gene sequence. The study reinforces the traditional concept that conservative interpretations of relatively low-resolution structures remain correct even with the availability of high-resolution data.

Degeneracy in the emergence of spike-triggered average of hippocampal pyramidal neurons.

Hippocampal pyramidal neurons are endowed with signature excitability characteristics, exhibit theta-frequency selectivity - manifesting as impedance resonance and as a band-pass structure in the spike-triggered average (STA) - and coincidence detection tuned for gamma-frequency inputs. Are there specific constraints on molecular-scale (ion channel) properties in the concomitant emergence of cellular-scale encoding (feature detection and selectivity) and excitability characteristics? Here, we employed a biophysically-constrained unbiased stochastic search strategy involving thousands of conductance-based models, spanning 11 active ion channels, to assess the concomitant emergence of 14 different electrophysiological measurements. Despite the strong biophysical and physiological constraints, we found models that were similar in terms of their spectral selectivity, operating mode along the integrator-coincidence detection continuum and intrinsic excitability characteristics. The parametric combinations that resulted in these functionally similar models were non-unique with weak pair-wise correlations. Employing virtual knockout of individual ion channels in these functionally similar models, we found a many-to-many relationship between channels and physiological characteristics to mediate this degeneracy, and predicted a dominant role for HCN and transient potassium channels in regulating hippocampal neuronal STA. Our analyses reveals the expression of degeneracy, that results from synergistic interactions among disparate channel components, in the concomitant emergence of neuronal excitability and encoding characteristics.

Comparative study of 7S globulin from Corylus avellana and Solanum lycopersicum revealed importance of salicylic acid and Cu-binding loop in modulating their function.

The plant seed proteins referred to as vicilins belong to a structurally common superfamily. While some of them are reported to exhibit superoxide dismutase activity, vicilins from other sources do not possess this activity. Vicilin from Corylus avellana (HZ.1) and Solanum lycopersicum (SL80.1) were purified and subjected to structure-function analysis. The superoxide dismutase activity assays were performed to understand the functional differences between them. While SL80.1 has the superoxide dismutase activity, HZ.1 was enzymatically inactive. Crystal structure followed by mass spectrometry analysis of both the proteins revealed that while SL80.1 has bound salicylic acid, HZ.1 does not. Comparison of C-terminal binding pocket of both the structures revealed that a point mutation at residue 321 in HZ.1 (Gly→Cys) leads to obstruction in binding of salicylic acid in the pocket. Similarly, copper-binding loop of HZ.1 was reportedly found to be intact and shorter than the loops reported in SL80.1. The copper-binding loop of SL80.1 is rich in polar residues and the absence of these residues in HZ.1 copper-binding loop possibly indicates deficiency in channeling of oxygen radicals to the active center of the enzyme. Difference in the enzymatic activity of vicilin from two evolutionarily distinct sources is due to mutations in its co-factor binding pocket and copper-binding loop.

Docking, thermodynamics and molecular dynamics (MD) studies of a non-canonical protease inhibitor, MP-4, from Mucuna pruriens.

Sequence and structural homology suggests that MP-4 protein from Mucuna pruriens belongs to Kunitz-type protease inhibitor family. However, biochemical assays showed that this protein is a poor inhibitor of trypsin. To understand the basis of observed poor inhibition, thermodynamics and molecular dynamics (MD) simulation studies on binding of MP-4 to trypsin were carried out. Molecular dynamics simulations revealed that temperature influences the spectrum of conformations adopted by the loop regions in the MP-4 structure. At an optimal temperature, MP-4 achieves maximal binding while above and below the optimum temperature, its functional activity is hampered due to unfavourable flexibility and relative rigidity, respectively. The low activity at normal temperature is due to the widening of the conformational spectrum of the Reactive Site Loop (RSL) that reduces the probability of formation of stabilizing contacts with trypsin. The unique sequence of the RSL enhances flexibility at ambient temperature and thus reduces its ability to inhibit trypsin. This study shows that temperature influences the function of a protein through modulation in the structure of functional domain of the protein. Modulation of function through appearance of new sequences that are more sensitive to temperature may be a general strategy for evolution of new proteins.

Crystal structure of nonspecific lipid transfer protein from Solanum melongena.

Lipids are considered to protect protein allergens from proteolysis and are generally seen to exist in a bound form. One of the well-known plant protein families with bound lipids is non-specific lipid transfer proteins (nsLTPs). Structure-function relationships in the case of the members of non-specific lipid transfer protein family are not clearly understood. As part of exploring the seed proteome, we have analyzed the proteome of a member of Solanaceae family, Solanum melongena (eggplant) and a non-specific lipid transfer protein from S. melongena, SM80.2 was purified, crystallized and the structure was determined at 1.87 Å resolution. Overall, the tertiary structure is a cluster of α-helices forming an internal hydrophobic cavity. Absence of conserved Tyr79, known to govern the plasticity of hydrophobic cavity, and formation of hydrogen bond between Asn79 and Asn36 further reduced the pocket size. Structural analysis of SM80.2 thus gives insight about a new hydrogen bond mediated mechanism followed in closure of the binding pocket. Extra electron densities observed at two different places on the protein surface and not in the cavity could provide interesting physiological relevance. In light of allergenic properties, probably overlapping of epitopic region and ligand binding on surface could be a main reason. This work shows first crystal structure of A-like nsLTP with a close binding pocket and extra density on the surface suggesting a plausible intermediate state during transfer.

Computational recognition and analysis of hitherto uncharacterized nucleotide cyclase-like proteins in bacteria.

UNLABELLED: Evolutionary relationship between class III nucleotide cyclases and an uncharacterized set of bacterial proteins from Actinobacteria, Bacteroidetes and Proteobacteria has been recognized and analyzed. Detailed analyses of sequence and structural features resulted in the recognition of potential cyclase function conferring residues and presence of signature topological motif (ßααßßαß) in the uncharacterized set of bacterial proteins. Lack of transmembrane domains and signal peptide cleavage sites is suggestive of their cytosolic subcellular localization. Furthermore, analysis on evolutionarily conserved gene clusters of the predicted nucleotide cyclase-like proteins and their evolutionary relationship with nucleotide cyclases suggest their participation in cellular signalling events. Our analyses suggest expansion of class III nucleotide cyclases. REVIEWERS: This article was reviewed by Eugene Koonin and Michael Gromiha.

Crystal structure of the vicilin from Solanum melongena reveals existence of different anionic ligands in structurally similar pockets.

Crystal structure of a vicilin, SM80.1, was determined towards exploring its possible physiological functions. The protein was purified from Solanum melongena by combination of ammonium sulphate fractionation and size exclusion chromatography. Structure was determined ab initio at resolution of 1.5 Å by X-ray crystallography showing the three-dimensional topology of the trimeric protein. Each monomer of SM80.1 consists of two similar domains with hydrophobic binding pocket and each accommodating different ligands, i.e. acetate and pyroglutamate. The relatively high stability of these independent anionic ligands in similar pockets indicated a strict requirement of stabilization by hydrogen bonds with the charged residues, suggesting a degree of plasticity within the binding pocket. Comparison of SM80.1 structure with those of other 7S vicilins indicated conservation of putative binding pocket for anionic ligands. Here we propose the possibility of trapping of these ligands in the protein for their requirement in the metabolic processes.

Purification, identification and preliminary crystallographic studies of an allergenic protein from Solanum melongena.

Solanum melongena (eggplant), a member of the Solanaceae family, is a widely cultivated vegetable crop and is commonly used as a food throughout the world. Allergic reactions caused by members of this family are well known. However, mechanistic analyses to understand their molecular basis have not been adequately explored. In order to address this issue, the 7S vicilin protein (SM80.1) of size 45 kDa was purified from seeds of S. melongena by ammonium sulfate fractionation and size-exclusion chromatography. Significant homology of SM80.1 to an allergy-related protein from S. lycopersicum was identified through a BLAST search. Crystallization attempts with purified protein using the hanging-drop vapour-diffusion method led to hexagonal-shaped crystals. The crystals diffracted to 2.21 Å resolution and belonged to space group P6322, with unit-cell parameters a = 117.9, c = 123.5 Å.

The structure of a haemopexin-fold protein from cow pea (Vigna unguiculata) suggests functional diversity of haemopexins in plants.

The haemopexin fold is present in almost all life forms and is utilized for carrying out diverse physiological functions. The structure of CP4, a haemopexin-fold protein from cow pea (Vigna unguiculata), was determined at 2.1 Šresolution. The protein exists as a monomer both in solution and in the crystal. The structure revealed a typical four-bladed ß-propeller topology. The protein exhibits 42% sequence similarity to LS-24 from Lathyrus sativus, with substantial differences in the surface-charge distribution and in the oligomeric state. A structure-based sequence analysis of haemopexin-fold proteins of plant and mammalian origin established a sequence signature associated with the haemopexin motif. This signature sequence enabled the identification of other proteins with possible haemopexin-like topology of both plant and animal origin. Although CP4 shares a structural fold with LS-24 and other haemopexins, biochemical studies indicated possible functional differences between CP4 and LS-24. While both of these proteins exhibit spermine-binding potential, CP4 does not bind to haem, unlike LS-24.

Explicit magnification control of self-organizing maps for "forbidden" data.

In this paper, we examine the scope of validity of the explicit self-organizing map (SOM) magnification control scheme of Bauer et al. (1996) on data for which the theory does not guarantee success, namely data that are n-dimensional, n > or =2, and whose components in the different dimensions are not statistically independent. The Bauer et al. algorithm is very attractive for the possibility of faithful representation of the probability density function (pdf) of a data manifold, or for discovery of rare events, among other properties. Since theoretically unsupported data of higher dimensionality and higher complexity would benefit most from the power of explicit magnification control, we conduct systematic simulations on "forbidden" data. For the unsupported n=2 cases that we investigate, the simulations show that even though the magnification exponent alpha achieved achieved by magnification control is not the same as the desired alpha desired, alpha achieved systematically follows alpha desired with a slowly increasing positive offset. We show that for simple synthetic higher dimensional data information, theoretically optimum pdf matching (alpha achieved = 1) can be achieved, and that negative magnification has the desired effect of improving the detectability of rare classes. In addition, we further study theoretically unsupported cases with real data.