The Ca2+-binding sequence in bovine brain S100b protein β-subunit. A spectroscopic study

Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.

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Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b

Biochemical Journal ◽

10.1042/bj2180045 ◽

1984 ◽

Vol 218 (1) ◽

pp. 45-60 ◽

Cited By ~ 20

Author(s):

A Lorek ◽

K S Wilson ◽

M S P Sansom ◽

D I Stuart ◽

E A Stura ◽

...

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Inorganic Phosphate ◽

Glycogen Phosphorylase ◽

Phosphate Binding ◽

Binding Studies ◽

Structural Explanation ◽

Alpha Helices ◽

Allosteric Effector ◽

Glycogen Phosphorylase B

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.

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Binding Studies of AICAR and Human Serum Albumin by Spectroscopic, Theoretical, and Computational Methodologies

Molecules ◽

10.3390/molecules25225410 ◽

2020 ◽

Vol 25 (22) ◽

pp. 5410

Author(s):

Shokoufeh Hashempour ◽

Nahid Shahabadi ◽

Aishat Adewoye ◽

Brennen Murphy ◽

Camaray Rouse ◽

...

Keyword(s):

Human Serum Albumin ◽

Fluorescence Quenching ◽

Serum Albumin ◽

Human Serum ◽

Binding Site ◽

Conformational Changes ◽

Flufenamic Acid ◽

Drug Candidate ◽

Protein Fluorescence ◽

Binding Studies

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR’s pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR–HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern–Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR–HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA’s binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR–HSA complexes, reaffirming that AICAR binds to both site I and site II.

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Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins

Science ◽

10.1126/science.aan6874 ◽

2017 ◽

Vol 358 (6369) ◽

pp. 1431-1434 ◽

Cited By ~ 49

Author(s):

Tea Lenarčič ◽

Isabell Albert ◽

Hannah Böhm ◽

Vesna Hodnik ◽

Katja Pirc ◽

...

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Pathogenic Bacteria ◽

Head Group ◽

Microbial Infection ◽

Binding Studies ◽

X Ray ◽

Plant Pathogenic Bacteria ◽

X Ray Crystallography ◽

Host Selectivity

Necrosis and ethylene-inducing peptide 1–like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

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Synthetic and binding studies on the calcium binding site I of bovine brain calmodulin

International journal of peptide & protein research ◽

10.1111/j.1399-3011.1985.tb01020.x ◽

2009 ◽

Vol 26 (5) ◽

pp. 528-538 ◽

Cited By ~ 15

Author(s):

G. BORIN ◽

A. PEZZOLI ◽

F. MARCHIORI ◽

E. PEGGION

Keyword(s):

Binding Site ◽

Calcium Binding ◽

Bovine Brain ◽

Binding Studies ◽

Calcium Binding Site

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5712

Author(s):

Michał Tracz ◽

Ireneusz Górniak ◽

Andrzej Szczepaniak ◽

Wojciech Białek

Keyword(s):

Ubiquitin Ligase ◽

Conformational Changes ◽

Protein Interactions ◽

E3 Ubiquitin Ligase ◽

Lanthanide Ions ◽

E3 Ligases ◽

Fluorescence Measurements ◽

Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

Biochemical Journal ◽

10.1042/bj3500485 ◽

2000 ◽

Vol 350 (2) ◽

pp. 485-493 ◽

Cited By ~ 1

Author(s):

Danny S. TUCKWELL ◽

Lyndsay SMITH ◽

Michelle KORDA ◽

Janet A. ASKARI ◽

Sentot SANTOSO ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Binding Site ◽

Conformational Changes ◽

Cation Binding ◽

Inhibitory Antibody ◽

Collagen Binding ◽

Collagen Peptide ◽

Binding Residue ◽

Important Region ◽

Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

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A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4256 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4256-4265 ◽

Cited By ~ 24

Author(s):

C J Brandl ◽

K Struhl

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Nucleosome Positioning ◽

Alternative Mechanism ◽

Tata Element ◽

Binding Factor ◽

Positioning Analysis ◽

Binding Sequence

In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element. This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction. Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA. One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site. Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription. The Q element specifically interacts with a yeast protein (Q-binding protein [QBP]) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning. Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter. These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA. In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.

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Engineered occluded apo-intermediate of LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816267115 ◽

2018 ◽

Vol 115 (50) ◽

pp. 12716-12721 ◽

Cited By ~ 1

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

H. Ronald Kaback

Keyword(s):

Binding Site ◽

Lactose Permease ◽

Binding Studies ◽

X Ray Crystallography ◽

Sugar Binding ◽

Transport Cycle ◽

Open Structures ◽

Unrestricted Access ◽

Tight Association ◽

Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

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Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

The Journal of General Physiology ◽

10.1085/jgp.201311148 ◽

2014 ◽

Vol 143 (4) ◽

pp. 449-464 ◽

Cited By ~ 45

Author(s):

Natascia Vedovato ◽

David C. Gadsby

Keyword(s):

Binding Site ◽

Single Molecule ◽

Conformational Changes ◽

Outward Current ◽

Side Chain ◽

Ion Binding ◽

Inward Current ◽

Na Release ◽

K Transport ◽

External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

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