N- and C-Terminal Domains of the Calcium Binding Protein EhCaBP1 of the Parasite Entamoeba histolytica Display Distinct Functions

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, and calcium signaling is thought to be involved in amoebic pathogenesis. EhCaBP1, a Ca2+ binding protein of E. histolytica, is essential for parasite growth. High resolution crystal structure of EhCaBP1 suggested an unusual arrangement of the EF-hand domains in the N-terminal part of the structure, while C-terminal part of the protein was not traced. The structure revealed a trimer with amino terminal domains of the three molecules interacting in a head-to-tail manner forming an assembled domain at the interface with EF1 and EF2 motifs of different molecules coming close to each other. In order to understand the specific roles of the two domains of EhCaBP1, the molecule was divided into two halves, and each half was separately expressed. The domains were characterized with respect to their structure, as well as specific functional features, such as ability to activate kinase and bind actin. The domains were also expressed in E. histolytica cells along with green fluorescent protein. The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase. Crystal structure of EhCaBP1 with Phenylalanine revealed that the assembled domains, which are similar to Calmodulin N-terminal domain, bind to Phenylalanine revealing the binding mode to the target proteins. The C-terminal domain did not show any of the activities tested. However, over-expression in amebic cells led to a dominant negative phenotype. The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other. Both the domains are required for structural stability and full range of functional diversity

Three-Dimensional Structure of N-Terminal Domain of DnaB Helicase and Helicase-Primase Interactions in Helicobacter pylori

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria

Axial and Radial Forces of Cross-Bridges Depend on Lattice Spacing

Nearly all mechanochemical models of the cross-bridge treat myosin as a simple linear spring arranged parallel to the contractile filaments. These single-spring models cannot account for the radial force that muscle generates (orthogonal to the long axis of the myofilaments) or the effects of changes in filament lattice spacing. We describe a more complex myosin cross-bridge model that uses multiple springs to replicate myosin's force-generating power stroke and account for the effects of lattice spacing and radial force. The four springs which comprise this model (the 4sXB) correspond to the mechanically relevant portions of myosin's structure. As occurs in vivo, the 4sXB's state-transition kinetics and force-production dynamics vary with lattice spacing. Additionally, we describe a simpler two-spring cross-bridge (2sXB) model which produces results similar to those of the 4sXB model. Unlike the 4sXB model, the 2sXB model requires no iterative techniques, making it more computationally efficient. The rate at which both multi-spring cross-bridges bind and generate force decreases as lattice spacing grows. The axial force generated by each cross-bridge as it undergoes a power stroke increases as lattice spacing grows. The radial force that a cross-bridge produces as it undergoes a power stroke varies from expansive to compressive as lattice spacing increases. Importantly, these results mirror those for intact, contracting muscle force production

PAVeDS: A Synthetic Dataset for Developing Autonomous Personal Aerial Vehicles

Aircraft designed for transporting one or two persons, much like a small personal car but flying in the air based on Bernoulli Principle and Newton’s laws, are called Personal Aerial Vehicles (PAVs). Due to high PAV traffic densities and the high velocities at which PAVs fly, manual piloting of PAVs is seldom recommended. Hence, PAVs are equipped with an inbuilt Autonomous Navigation and Control System (ANCS), which frees the rider from piloting skills. Machine learning (ML)-based approaches that require datasets used during training phases can be used for implementing the software of such ANCS. The development of simulation-driven systems for ANCS offers many advantages, particularly reducing the systems’ developmental costs and infrastructure needs. In this article, we report on the development of a synthetic visual dataset that enables ML-based implementation of ANCS. The state-of-the-art simulator AirSim is used to generate this dataset. Additionally, to make the synthetic data more realistic, several augmenting technologies such as Unreal Engine (3D graphics gaming), Blender animator, PX4-Autopilot SITL (Software in the loop) software, QGroundControl Autopilot App, and ROS (robot operating system) middleware suite are utilized. We also discuss the applicability of this dataset in realizing the ANCS module for PAVs

Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported

The binding free energy of EhSAT1 and EhSAT3 complexes.

<p>The table shows the detailed contribution of energy components calculated using Poisson Boltzmann Surface Area (MM-PBSA) method for EhSAT1, EhSAT3 and their mutants to evaluate their binding activity. Here ΔE_Ele, electrostatic interactions; ΔE_Vdw, van der Waals interactions, ΔE_MM = ΔE _Ele+ΔE_Vdw, ΔG_sol-ele: polar solvation free energy are calculated by solving the Poisson-Boltzmann equation PB; ΔG_sol-np, non-polar solvation free energy, ΔG_polar = ΔE _Ele+ΔG_sol-ele; ΔG_nonpolar = ΔE_Vdw+ΔG_sol-np, ΔG_Bind = estimated total binding free energy.</p

Sequence and crystal structure determination of a basic phospholipase A2 from common krait (Bungarus caeruleus) at 2.4 Å resolution: identification and characterization of its pharmacological sites1

This is the first phospholipase A2 (PLA2) structure from the family of kraits. The protein was isolated from Bungarus caeruleus (common krait) and the primary sequence was determined using cDNA approach. Three-dimensional structure of this presynaptic neurotoxic PLA2 from group I has been determined by molecular replacement method using the model of PLA2 component of β2-bungarotoxin (Bungarus multicinctus) and refined using CNS package to a final R-factor of 20.1 % for all the data in resolution range 20.0-2.4 Å. The final refined model comprises 897 protein atoms and 77 water molecules. The overall framework of krait phospholipase A2 with three long helices and two short antiparallel β-strands is extremely similar to those observed for other group I PLA2s. However, the critical parts of PLA2 folding are concerned with its various functional loops. The conformations of these loops determine the efficiency of enzyme action and presence/absence of various pharmacological functions. In the present structure calcium-binding loop is occupied by a sodium ion with a 7-fold co-ordination. The conformation of loop 55-75 in krait PLA2 corresponds to a very high activity of the enzyme. A comparison of its sequence with multimeric PLA2s clearly shows the absence of critical residues such as Tyr3, Trp61 and Phe64, which are involved in the multimerization of PLA2 molecules. The protein shows anticoagulant and neurotoxic activities

Protein sequence alignment of EhSAT1 and EhSAT3.

<p>Protein sequence alignment was done using ClustalW alignment program. Conserved active site residues are highlighted in yellow boxes while the active site residues (at position 208) that differ in the isoforms are highlighted in a pink box.</p

Structure of the bifunctional inhibitor of trypsin and α-amylase from ragi seeds at 2.2 Å resolution

The crystal structure of a bifunctional inhibitor of α-amylase and trypsin (RATI) from ragi seeds (Indian finger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2Å resolution. The inhibitor consists of 122 amino acids, with five disulfide bridges, and belongs to the plant α-amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 Å resolution determined previously. It has been refined to an R factor of 21.9%. The structure shows an r.m.s. deviation for C atoms of 2.0 Åcompared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 Å. The r.m.s. difference for C atoms when compared with the same protein in the structure of the complex with α-amylase is 0.7 Å. The conformations of trypsin-binding loop and the -amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with α-amylase. These regions differed considerably in the NMR structure

Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from <em>Entamoeba histolytica</em> Assists in Partial Regaining of Feedback Inhibition by Cysteine

<div><p>The cysteine biosynthetic pathway is essential for survival of the protist pathogen <i>Entamoeba histolytica,</i> and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated <i>in vitro</i>, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by K_m, compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3) shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor.</p> </div