SignificanceThe preparation of membrane-protein specimens for structural characterization commonly involves a detergent-solubilization step that is sometimes followed by the reincorporation of the protein into laboratory-made lipid nanodiscs. Whether the native conformation and arrangement of tightly bound lipids are retained despite solubilization or restored upon nanodisc formation cannot be predicted. To determine the structure of the bacterial pentameric ligand-gated ion channel ELIC in its native environment, we overexpressed it inEscherichia coli, fragmented the inner membranes into disc-shaped structures in the absence of detergents, purified discs containing single copies of the protein, and imaged the resulting particles using cryogenic electron microscopy. The obtained three-dimensional reconstruction of the unliganded channel (2.5-Å resolution) revealed previously unseen details of the lipid–protein interface.

The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC ( Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C—at the agonist-binding sites—and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD–TMD coupling in such a way that the “conformational wave” of liganded-receptor gating takes place in the ECD and the interfacial M2–M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand’s fluctuations, and thus delays its escape into bulk solvent.

2S albumin is a low-molecular-weight seed storage protein belonging to the prolamin superfamily. In the present work a small 2S albumin (WTA) protein of ~16 kDa has been purified from the seeds of Wrightia tinctoria. The WTA is a heterodimer protein with a small subunit of ~5 kDa and a larger subunit of ~11 kDa bridged together through disulphide bonds. The protein exhibits deoxyribonucleases activity against closed circular pBR322 plasmid DNA and linear BL21 genomic DNA. The protein also showed antibacterial activity against Morexalla catarrhalis. CD studies indicate a high α-helical content in the protein. The conserved disulphide bonds in the protein suggest that the WTA is highly stable under high pH and temperature like other 2S albumin.

Auxin levels are tightly regulated within the plant cell, and its storage in the isolated cavity of proteins is a measure adopted by cells to maintain the availability of auxin. We report the first crystal structure of Wrightia tinctoria 11S globulin (WTG) in complex with Indole-3-acetic acid (IAA), an auxin, at 1.7 Å resolution. WTG hexamers assemble as a result of the stacking interaction between the hydrophobic surfaces of two trimers, leaving space for the binding of charged ligands. The bound auxin is stabilized by non-covalent interactions, contributed by four chains in each cavity. The presence of bound ligand was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-resolution mass spectrometry (HRMS). Here, we hypothesize that the cleavage of globulins by endopeptidases leads to the movement of the hydrophilic loop region from the surface to the periphery, leaving space for the binding of auxin, and promotes hexamer formation. As the process of germination proceeds, there is a change in the pH, which induces the dissociation of the hexamer and the release of auxin. The compact hexameric assembly ensures the long-term, stable storage of the hormone. This suggests a role for globulin as a novel player in auxin homeostasis.

Globulins are a major class of seed storage proteins which were thought to be enzymatically inactive. These proteins belong to the most ancient cupin superfamily. They can be graded into 11S legumin type and 7S vicilin type based on their sedimentation coefficients. Members from both classes share structural homology are thought to have evolved from either one-domain germin predecessor by duplication or by horizontal gene transfer of two-domain gene from bacteria to eukaryotes. Globulins are known to define the nutritional quality of the seeds, however, they are also involved in sucrose binding, desiccation, defense against microbes, hormone binding and oxidative stress etc. Major drawback with globulins is their tendency to bind to IgE. Studying structural-functional behavior of such protein can help in modifying proteins for enhanced functionality in food processing industries.

The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure-function evolution of CLPs from chitinases.

The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase‐like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure–function evolution of CLPs from chitinases.

KdsC, the third enzyme of the 3-deoxy-d-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-d-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC from Moraxella catarrhalis (Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for the Mc-KdsC enzyme.

Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS-PAGE gel under non-reducing conditions, a major band of similar to 56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight similar to 32-34 kDa and the other with molecular weight similar to 22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of beta-sheets (44.2%) and moderate content of alpha-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 degrees C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of alpha=109.9 angstrom, b=113.2 angstrom and c=202.2 angstrom with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2 angstrom under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.

This paper outlines a mathematical model developed for steady-state multistage completely mixed hybrid reactors, starting from basic mass balance expressions for the system and Fick's theory for diffusion of substrate into the biofilm. The model being complex, a design procedure has been proposed that uses predefined effluent quality profile across the system and results in an accurate and optimum design of the system for a desired degree of treatment. Using reported values of kinetic parameters from literature, it has been concluded that the proposed computationally efficient procedure can be successfully used for the design and control of these reactors.