Structural insights intoAspergillus fumigatuslectin specificity: AFL binding sites are functionally non-equivalent

2015 ◽  
Vol 71 (3) ◽  
pp. 442-453 ◽  
Author(s):  
Josef Houser ◽  
Jan Komarek ◽  
Gianluca Cioci ◽  
Annabelle Varrot ◽  
Anne Imberty ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Opportunistic Pathogen ◽  
Biotechnological Applications ◽  
Binding Properties ◽  
High Affinity ◽  
Lectin Family ◽  
Structural Insights ◽  
Specific Ligand ◽  
Micromolar Range

TheAspergillus fumigatuslectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications.

scholarly journals Computational Insights into the Interaction between Cytoadherence Receptor gC1qR and the DBLβ12 Domain of a Plasmodium falciparum PfEMP1 Ligand

Life ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 993
Author(s):  
Rowaida Bakri ◽  
Mohd Rehan ◽  
Hina Shamshad ◽  
Abdul Hafiz
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Binding Sites ◽  
Molecular Model ◽  
Protein Docking ◽  
Amino Acid Residues ◽  
Structural Insights ◽  
Malaria Interventions ◽  
Falciparum Erythrocyte Membrane Protein ◽  
Human Receptor

Human receptor gC1qR is a 32 kD protein that mediates the cytoadherence of Plasmodium falciparum-infected erythrocytes (IEs) to human brain microvascular endothelial cells (HBMEC) and platelets. The cytoadherence of IEs to gC1qR has been associated with severe malaria symptoms. The cytoadherence to gC1qR is mediated by the Duffy binding-like β12 (DBLβ12) domain of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), PFD0020c. Here, we report the structural insights into the binding of the DBLβ12 domain of PfEMP1 with the human receptor gC1qR using computational methods. A molecular model of the DBLβ12 domain was generated and used for protein–protein docking with the host receptor gC1qR. The protein–protein docking revealed that the DBLβ12 asymmetrically interacts with two subunits of the gC1qR trimer at the solution face of gC1qR. A total of 21 amino acid residues of DBLβ12 interact with 26 amino acid residues in the gC1qR trimer through 99 nonbonding interactions and 4 hydrogen bonds. Comparative analysis of binding sites on the DBL domain fold for the two receptors gC1qR and ICAM1 showed that the two sites are distinct. This is the first study that provides structural insights into DBLβ12 binding with its receptor gC1qR and may help in designing novel antisevere malaria interventions.

scholarly journals Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs inPseudomonas aeruginosa

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Matthew Golden ◽  
Justin W. Graham ◽  
Wes Sanders ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Opportunistic Pathogen ◽  
Binding Properties ◽  
Content Type ◽  
Preferential Binding ◽  
Regulatory Rnas

ABSTRACTPseudomonas aeruginosais a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCECsrA homologs are present in many bacteria. The opportunistic pathogenPseudomonas aeruginosauses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activityin vivo. Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activityin vivoandin vitroare more stringent than those for RsmA.

scholarly journals Alternative ligands as probes for the carotenoid-binding site of lobster carapace crustacyanin

1990 ◽  
Vol 265 (3) ◽  
pp. 919-921 ◽  
Author(s):  
J B Clarke ◽  
E E Eliopoulos ◽  
J B C Findlay ◽  
P F Zagalsky
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Binding Sites ◽  
Amino Acid Sequences ◽  
Fluorescence Probes ◽  
Affinity Binding ◽  
Hydrophobic Pocket ◽  
High Affinity Binding ◽  
High Affinity ◽  
Parinaric Acid

The apoproteins of the lobster carotenoprotein, crustacyanin, show single high-affinity binding sites for the hydrophobic fluorescence probes 8-anilo-1-naphthalenesulphonic acid and cis-parinaric acid, and exhibit fluorescence transfer from tryptophan to the ligands. These results, together with information from the amino acid sequences, infer that the native carotenoid, astaxanthin, is bound to each apoprotein within an internal hydrophobic pocket, or calyx.

scholarly journals Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

2015 ◽  
Vol 112 (6) ◽  
pp. 1755-1760 ◽  
Author(s):  
Mette Laursen ◽  
Jonas Lindholt Gregersen ◽  
Laure Yatime ◽  
Poul Nissen ◽  
Natalya U. Fedosova
Keyword(s):  
Binding Sites ◽  
Cation Binding ◽  
General Mechanism ◽  
Transmembrane Helices ◽  
Binding Properties ◽  
High Affinity ◽  
Cation Binding Site ◽  
Fine Tune ◽  
Specific Influence

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na+,K+-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na+,K+-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P–ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg2+ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K+ in the E2P–bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1–2.

scholarly journals DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context.

1988 ◽  
Vol 8 (11) ◽  
pp. 4598-4607 ◽  
Author(s):  
T Hoey ◽  
R Warrior ◽  
J Manak ◽  
M Levine
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Binding Properties ◽  
Binding Behavior ◽  
High Affinity ◽  
Homeo Box ◽  
Acid Protein ◽  
Dna Binding Properties ◽  
Amino Acid Protein

The homeo box gene even-skipped (eve) encodes a 376-amino-acid protein that binds with high affinity to sequences located near the 5' termini of the eve and en genes. The 5' en sites are A + T rich and contain copies of the 10-base-pair (bp) consensus sequence T-C-A-A-T-T-A-A-A-T. In contrast, the 5' eve sites are G + C rich and contain the 9-bp sequence T-C-A-G-C-A-C-C-G. Among the five different homeo box proteins that have been tested for binding, eve is unique in that it shows virtually equal preference for the A + T-rich 5' en binding sites and the G + C-rich 5' eve sites. Most of the other proteins bind with a relatively higher affinity to the en sites than to the eve sites. In an effort to identify the regions of the eve protein that are responsible for its efficient binding to both classes of recognition sequences, we analyzed the DNA-binding properties of various mutant eve proteins. These studies suggest that the homeo domain of the eve protein is responsible for both binding activities. However, mutations in distant regions of the protein influenced the binding behavior of the eve homeo domain and caused a reduction in binding to the G + C class of recognition sites. We propose that the protein context of the homeo domain can influence its DNA-binding properties.

scholarly journals Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

2021 ◽  
Author(s):  
Judy L.M. Kotler ◽  
Wei-Shao Wei ◽  
Erin E Deans ◽  
Timothy O. Street
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Client Protein ◽  
High Affinity ◽  
Hsp70 Family ◽  
Differential Affinity ◽  
Peptide Region ◽  
Positively Charged ◽  
Micromolar Range

Hsp70-family chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how Hsp70s differentiate between monomeric and oligomeric states of a target protein. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. We discover that electrostatic attraction enables the negatively charged BiP to bind positively charged E-peptide oligomers with low nanomolar affinity. We identify the specific BiP binding sites on proIGF2, and although some are positively charged, as monomers they bind BiP with characteristically low affinity in the micromolar range. We conclude that electrostatics enable BiP to preferentially recognize oligomeric states of proIGF2. Electrostatic targeting of Hsp70 to aggregates may be broadly applicable, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that are expected to be positively charged.

scholarly journals A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1986 ◽  
Vol 237 (1) ◽  
pp. 217-227 ◽  
Author(s):  
G W Gould ◽  
J M East ◽  
R J Froud ◽  
J M McWhirter ◽  
H I Stefanova ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Affinity ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Single Binding Site ◽  
Micromolar Range

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1′PCa2 and to E2 modulate the rates of the transport step E1′PCa-E2′PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.

scholarly journals Preparation of the alkali and P light chains of chicken gizzard myosin. Amino acid sequence of the alkali light chain

1983 ◽  
Vol 211 (1) ◽  
pp. 267-272 ◽  
Author(s):  
R J A Grand ◽  
S V Perry
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Light Chain ◽  
Binding Sites ◽  
Light Chains ◽  
British Library ◽  
Simple Method ◽  
Chicken Gizzard ◽  
High Affinity

1. A simple method is described for the purification of the alkali and P light chains from chicken gizzard myosin. 2. The sequence of the alkali light chain has been unequivocally determined, except for the N-terminal dipeptide, by using the tryptic and CNBr peptides. 3. No evidence was obtained for any specific high-affinity Ca2+-binding sites on the alkali light chain. 4. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50120 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1983) 209, 5.

Renal receptors for atrial and C-type natriuretic peptides in the rat

1992 ◽  
Vol 263 (1) ◽  
pp. F89-F96 ◽  
Author(s):  
J. Brown ◽  
Z. Zuo
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Binding Sites ◽  
Wistar Rats ◽  
Apparent Dissociation Constant ◽  
High Affinity ◽  
Vasa Recta ◽  
Dissociation Constant Kd ◽  
Specific Ligand

Receptors for alpha-atrial natriuretic peptide (alpha-ANP) and C-type natriuretic peptide [CNP-(1-22)] were quantified in kidneys from adult Wistar rats by in vitro autoradiography. 125I-labeled alpha-ANP (100 pM) bound reversibly to glomeruli, outer medullary vasa recta, and inner medulla with an apparent dissociation constant (Kd) of 3–6 nM. The presence of 10 microM des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4– 23) (C-ANP), a specific ligand of the ANPR-C subtype of alpha-ANP receptor, inhibited approximately 50% of the glomerular binding of 125I-alpha-ANP, and this moiety of glomerular binding was also inhibited by CNP-(1–22) with an apparent inhibitory constant (Ki) of 10.47 +/- 7.59 nM. C-ANP and CNP-(1–22) showed little affinity for the medullary binding sites of alpha-ANP. 125I-[Tyr0]CNP-(1–22) (110 pM) bound solely to glomeruli and was competitively displaced by increasing concentrations of [Tyr0]CNP-(1–22) with an apparent Kd of 1.42 +/- 0.48 nM. Binding of increasing concentrations (25 pM to 1 nM) of 125I-[Tyr0]CNP-(1–22) in the presence or absence of 1 microM [Tyr0]CNP-(1–22) also demonstrated a high affinity (Kd of 0.41 +/- 0.07 nM) for the glomerular binding of 125I-[Tyr0]CNP-(1–22). Bound 125I-[Tyr0]CNP-(1–22) could be displaced by excess alpha-ANP and excess CNP-(1–22), both with high affinities. The glomerular binding of 125I-[Tyr0]CNP-(1–22) was also prevented by 10 microM C-ANP. Guanosine 3',5'-cyclic monophosphate produced by isolated glomeruli was measured by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

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