Role Of His Research Articles

Histidine-Tagged Folate-Targeted Gold Nanoparticles for Enhanced Transgene Expression in Breast Cancer Cells In Vitro.

Nanotechnology has emerged as a promising treatment strategy in gene therapy, especially against diseases such as cancer. Gold nanoparticles (AuNPs) are regarded as favorable gene delivery vehicles due to their low toxicity, ease of synthesis and ability to be functionalized. This study aimed to prepare functionalized AuNPs (FAuNPs) and evaluate their folate-targeted and nontargeted pCMV-Luc-DNA delivery in breast cancer cells in vitro. CS was added to induce stability and positive charges to the AuNPs (Au-CS), histidine (Au-CS-His) to enhance endosomal escape and folic acid for folate-receptor targeting (Au-CS-FA-His). The FAuNP:pDNA nanocomplexes possessed favorable sizes (<135 nm) and zeta potentials (<−20 mV), strong compaction efficiency and were capable of pDNA protection against nuclease degradation. These nanocomplexes showed minimal cytotoxicity (>73% cell viability) and enhanced transgene activity. The influence of His was notable in the HER2 overexpressing SKBR3 cells, which produced higher gene expression. Furthermore, the FA-targeted nanocomplexes enhanced receptor-mediated endocytosis, especially in MCF-7 cells, as confirmed by the receptor competition assay. While the role of His may need further optimization, the results achieved suggest that these FAuNPs may be suitable gene delivery vehicles for breast cancer therapeutics.

A study on the secondary structure of the metalloregulatory protein CueR: effect of pH, metal ions and DNA.

The response of CueR towards environmental changes in solution was investigated. CueR is a bacterial metal ion selective transcriptional metalloregulator protein, which controls the concentration of copper ions in the cell. Although several articles have been devoted to the discussion of the structural and functional features of this protein, CueR has not previously been extensively characterized in solution. Here, we studied the effect of change in pH, temperature, and the presence of specific or non-specific binding partners on the secondary structure of CueR with circular dichroism (CD) spectroscopy. A rather peculiar reversible pH-dependent secondary structure transformation was observed, elucidated and supplemented with pKa estimation by PROPKA and CpHMD simulations suggesting an important role of His(76) and His(94) in this process. CD experiments revealed that the presence of DNA prevents this structural switch, suggesting that DNA locks CueR in the α-helical-rich form. In contrast to the non-cognate metal ions HgII, CdII and ZnII, the presence of the cognate AgI ion affects the secondary structure of CueR, most probably by stabilizing the metal ion and DNA-binding domains of the protein.

A Study on the Role of HIS for Effective Work Utilization in Hotel Industry

A Study on the Role of HIS for Effective Work Utilization in Hotel Industry

Hudjwiri and the role of HIS “Kashf UL-Makhjoob” in the Sufi History

Hudjwiri and the role of HIS “Kashf UL-Makhjoob” in the Sufi History

Gating role of His 72 in TmPurL enzyme uncovered by structural analyses and molecular dynamics simulations

Histidine is ubiquitous in enzyme active sites but its role is often difficult to ascribe due to ambiguity of protonation state and complex electrostatic and dynamic effects involved. In this study the role of His 72 in TmPurL, a glutamine amidotransferase (GAT) enzyme, is investigated. TmPurL is a large 66kDa enzyme that works as part of an even larger (>100kDa) multi-protein complex. This enzyme complex performs an essential step in the purine biosynthesis pathway by abstracting ammonia from a glutamine molecule and channeling it 30Å away into the active site of TmPurL, incorporating it into a purine biosynthesis intermediate. It is known that His 72 is important for substrate binding and possibly acts as a general base. Comparing apo and holo structural forms of this enzyme has revealed to us a possible gating function of His 72 that could regulate ammonia entry into the active site. Bimodal distribution of the χ1 dihedral angle of this amino acid in molecular dynamics simulations of 2μs supports the hypothesis. Different protonation states of His 72 were found to be conformationally distinct, providing a possible link between catalytic and gating roles of His 72. Ammonia channeling and allostery are discussed for GATs and more specifically for PurL family.

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of l-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme l-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA·FADH2 and of the ternary VioA·FADH2·2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of l-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of l-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using l-tryptophan and substrate analogs) identified Arg(64), Lys(269), and Tyr(309) as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of l-phenylalanine indicated a functional role of His(163) in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.

A novel synthetic derivative of melatonin, 5-hydroxy-2’-isobutyl-streptochlorin (HIS), inhibits inflammatory responses via regulation of TRIF-dependent signaling and inflammasome activation

Melatonin is substantially reported to possess anti-inflammatory properties. In the present study, we synthesized a novel melatonin derivative, 5-hydroxy-2′-isobutyl-streptochlorin (HIS), which displayed superior anti-inflammatory properties to its parent compound. Further, we explored its underlying mechanisms in cellular and experimental animal models. Lipopolysaccharide was used to induce in vitro inflammatory responses in RAW 264.7 macrophages. LPS-primed macrophages were pulsed with biologically unrelated toxic molecules to evaluate the role of HIS on inflammasome activation. In vivo verifications were carried out using acute lung injury (ALI) and Escherichia coli-induced septic shock mouse models. HIS inhibited the production of proinflammatory mediators and cytokines such as nitric oxide, cyclooxygenase 2, IL-1β, IL-6 and TNF-α in LPS-stimulated RAW 264.7 macrophages. HIS suppressed the infiltration of immune cells into the lung and the production of pro-inflammatory cytokines such as IL-6 and TNF-α in broncho-alveolar lavage fluid in the ALI mouse model. Mechanistic studies revealed that the inhibitory effects of HIS were mediated through the regulation of the TIR domain-containing, adaptor-inducing, interferon-β (TRIF)-dependent signaling pathway from toll-like receptors. Further, HIS attenuated IL-1β secretion via the inhibition of NLRP3 inflammasome activation independent of mitochondrial ROS production. Furthermore, HIS suppressed IL-1β, IL-6 and interferon-β production in peritoneal lavage in the Escherichia coli-induced sepsis mouse model. In conclusion, HIS exerted potent anti-inflammatory effects via the regulation of TRIF-dependent signaling and inflammasome activation. Notably, the superior anti-inflammatory properties of this derivative compared with its parent compound could be a promising lead for treating various inflammatory-mediated diseases.

Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM−1s−1 for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM−1s−1 for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235—located on a wide open groove near subsite +1—is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.

Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis.

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His(284) in the active site replaced by Asn, Asp or Tyr showed up to 10(4)-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His(284) in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.

Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1

SummaryThe NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in eukaryotes and bacteria through the uptake of peptides. However, in the majority of plant species members of this family have evolved to transport nitrate as well as additional secondary metabolites and hormones. In response to falling nitrate levels, NRT1.1 is phosphorylated on an intracellular threonine that switches the transporter from a low to high affinity state. Here we present both the apo and nitrate bound crystal structures of Arabidopsis thaliana NRT1.1, which together with in vitro binding and transport data identify a key role for His356 in nitrate binding. Our data support a model whereby phosphorylation increases structural flexibility and in turn the rate of transport. Comparison with peptide transporters further reveals how the NRT1/PTR family has evolved to recognize diverse nitrogenous ligands, whilst maintaining elements of a conserved coupling mechanism within this superfamily of nutrient transporters.

The Critical Role of Locally Conducted Research in Guiding the Response to the HIV Epidemic in Jamaica

Locally conducted research has played a critical role in guiding the response to the human immunodeficiency virus (HIV) epidemic in Jamaica. Active HIV/AIDS case-based surveillance and serosurveys helped to define the acquired immunodeficiency syndrome (AIDS) epidemic in Jamaica and identify those who were most at risk. Studies among sexually transmitted infection (STI) clinic attendees ident fled genital ulcers and syphilis as risk factors for HIV infection and were suggestive of other risk factors. Given the synergistic role of HI V and other STI, an integrated approach was taken to HIV/STI control and the STI services were strengthened based on research. Studies were conducted among sex-workers (SW), men who have sex with men, persons who go to sites to meet new sex partners, prisoners and others most at risk in order to understand risk factors and better guide the HIV/STI response. National population based surveys were conducted periodically to monitor knowledge, attitudes, beliefs as well as sexual behaviour and condom use. The research contributed greatly to Jamaica's comprehensive HIV/STI control programme that has been effective in slowing the HIV epidemic, reducing HlVprevalence among SW and STI clinic attendees, preventing mother-to-child transmission, reducing syphilis rates and mitigating the impact of HIV on the population.

Probing the catalytic mechanism of a C-3′-methyltransferase involved in the biosynthesis of D-tetronitrose

D-Tetronitrose is a nitro-containing tetradeoxysugar found attached to the antitumor and antibacterial agent tetrocarcin A. The biosynthesis of this highly unusual sugar in Micromonospora chalcea requires 10 enzymes. The fifth step in the pathway involves the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the C-3' carbon of dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose. The enzyme responsible for this transformation is referred to as TcaB9. It is a monomeric enzyme with a molecular architecture based around three domains. The N-terminal motif contains a binding site for a structural zinc ion. The middle- and C-terminal domains serve to anchor the SAM and dTDP-sugar ligands, respectively, to the protein, and the active site of TcaB9 is wedged between these two regions. For this investigation, the roles of Tyr 76, His 181, Tyr 222, Glu 224, and His 225, which form the active site of TcaB9, were probed by site-directed mutagenesis, kinetic analyses, and X-ray structural studies. In addition, two ternary complexes of the enzyme with bound S-adenosyl-L-homocysteine and either dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose or dTDP-3-amino-2,3,6-trideoxy-D-galactose were determined to 1.5 or 1.6 Å resolution, respectively. Taken together, these investigations highlight the important role of His 225 in methyl transfer. In addition, the structural data suggest that the methylation reaction occurs via retention of configuration about the C-3' carbon of the sugar.

Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed β-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 Å resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel β-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed β-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH-rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH-rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.

Structural and Mechanistic Insights into Unusual Thiol Disulfide Oxidoreductase

Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.

Molecular Architecture of TylM1 from Streptomyces fradiae: An N,N-Dimethyltransferase Involved in the Production of dTDP-d-mycaminose,

d-Mycaminose is an unusual dideoxy sugar found attached to the antibiotic tylosin, a commonly used veterinarian therapeutic. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar. The last step in its biosynthesis involves the dimethylation of the hexose C-3' amino group by an S-adenosylmethionine (SAM) dependent enzyme referred to as TylM1. Here we report two high-resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosylhomocysteine (SAH) and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 and 1.79 Å resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-3' amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of SAM. Both Tyr 14 and Arg 241 serve to anchor the dTDP-linked sugar to the protein. To test the role of His 123 in catalysis, two site-directed mutant proteins were constructed, H123A and H123N. Both mutant proteins retained catalytic activity, albeit with reduced rates. Specifically, the k(cat)/K(m) was reduced to 1.8% and 0.37% for the H123A and H123N mutant proteins, respectively. High-resolution X-ray models showed that the observed perturbations in the kinetic constants were not due to major changes in their three-dimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds.

A bipartite S unit of an ECF-type cobalt transporter

ECF-class transporters comprise abundant importers for micronutrients such as vitamins and transition-metal ions, and for intermediates of salvage pathways in bacteria and archaea. They are composed of ABC ATPases (A units), a conserved transmembrane protein (T unit) and a substrate-specific transmembrane protein (S unit or core transporter). Here we analyzed the function of an ECF-type Co 2+ transporter (CbiMNQO) and, in particular, the derived bipartite S unit CbiMN. CbiMN was characterized as the minimal unit that functions as a Co 2+ transporter. Neither the solitary CbiM nor a tripartite CbiMQO complex was active, indicating an essential role for CbiN. CbiN was loosely bound in CbiMNQO and CbiMN complexes, and did not copurify with its partners. Generating a contiguous reading frame resulted in a Cbi(MN) fusion protein that displayed Co 2+-transport activity and interacted with CbiQO in vivo. Sixteen variants of Cbi(MN) with modifications in the strongly conserved N-terminal stretch of ten amino-acid residues were constructed and analyzed for transport activity. The results indicate that the length and sequence of this region are critical for functioning of the core transporter. Specifically, they point to essential roles of His 2 and the distance of His 2 to the amino group of the peptide chain in metal recognition.

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca(2+) channel TRPV5 constitutes the apical entry gate for Ca(2+) transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca(2+) reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)) using fura-2 analysis. Removal of amino acid His(712) elevated the [Ca(2+)](i), indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His(712) for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His(712) was confirmed by patch clamp analysis, which demonstrates increased Na(+) and Ca(2+) currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca(2+)-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca(2+)](i). Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His(712) plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His 600. In the present work, the role of His 600 of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S 1 and S 1′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His 600 residue makes important interactions with the substrate, supporting the prediction that His 600 moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K m and k cat, showing the importance of His 600 for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His 600 in TOP catalysis, transferring a proton to the newly generated NH 2-terminus or helping Tyr 605 and/or Tyr 612 in the intermediate oxyanion stabilization.

Mutation of His 834 in human anion exchanger 1 affects substrate binding

Anion exchanger 1 (AE1 or band 3) is responsible for Cl(-)-HCO3(-) exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829-Phe 836) in yeast cell membranes. The K(m) values of the His 834 mutants were 4-10 times higher than that of the wild type, while their V(max) values were barely lower than that of wild type. Meanwhile, the K(m) values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding.

Role of His(E7) in Regulating Ligand Binding to the Subunits of Human HbA

To resolve previous discrepancies between structural and functional studies, the role of the distal histidine in HbA was re-evaluated by preparing Gly, Ala, Leu, Gln, Phe and Trp(E7) mutants and measuring the effects on O2, CO, and NO binding to mutant/wild type hybrid tetramers and isolated mutant subunits. Substituting His(E7) with apolar amino acids dramatically increases O2 dissociation (20-500-fold) in both subunits, suggesting equally strong hydrogen bonds between His(E7) and bound O2 (ΔGH-bond ≈ −5.6 kJ/mol). Increasing the size of the E7 residue from Gly to Phe results in monotonic decreases in the bimolecular rates of ligand binding to both subunits, supporting the E7 gate as the pathway for ligand entry in HbA. The results for the Trp(E7) mutants are more complex. Both fast (∼150-200 μM−1s−1) and one or more slow phases (1 to 0.1 μM−1s−1) are observed after photolysis of CO. The fraction of the fast phase decreases markedly when [CO] is lowered. In contrast, when isolated α and βTrp(E7) deoxyHb subunits are mixed with CO in stopped flow experiments, only slow phases are observed. Thus, after photolysis of the CO form of Trp(E7) mutants, there appears to be a competition between bimolecular ligand rebinding to an “open” conformation and the movement of the indole side chain back into the E7 channel forming an equilibrium “closed,” slowly reacting conformation. This mechanism is supported by the crystal structure of the CO form of α (wt)/βTrp(E7), in which the mutant indole side chain is in an open conformation exposed to solvent. In the deoxyHb crystal structure of αTrp(E7)/β (wt), the indole ring of Trp(E7) is in a closed conformation, blocking both the ligand binding site and the E7 channel for ligand entry.