Thermodynamic and Structural Studies of Carbohydrate Binding by the Agrin-G3 Domain

Sallum, Christine O.; Kammerer, Richard A.; Alexandrescu, Andrei T.

doi:10.1021/bi7006383

Cited by 8 publications

(4 citation statements)

References 63 publications

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“…ITC measurements for the ligands 9 k, 13 a,b, and 17 a-c were performed at 25 8C using hCD22 d1-3 -Fc [34] (Table 4) and confirmed the behavior typically observed for carbohydrate-lectin interactions, [40][41][42][43] namely an enthalpydriven binding. Less common are entropy-driven carbohydrate-lectin interactions, such as heparin binding to the agrin-G3 domain [42] or the interaction of di-and trisaccharides with calreticulin. [43] The interaction of the parent compound 9 k is dominated by a large enthalpic contribution, which is, however, decreased by substantial entropy costs (Entry 23).…”

Section: Isothermal Titration Calorimetry (Itc)supporting

confidence: 64%

From a Library of MAG Antagonists to Nanomolar CD22 Ligands

et al. 2011

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Siglec-2, also known as CD22, is involved in the regulation and survival of B-cells and has been successfully targeted in cell depletion therapies with antibody-based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin-associated glycoprotein (MAG, Siglec-4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2-, 5-, and 9-positions of the sialic acid scaffold, leading to nanomolar derivatives, of which ligand 17 b shows the most promising pharmacodynamic and pharmacokinetic profiles. Isothermal titration calorimetry measurements demonstrate that the binding is enthalpy driven. Interestingly, the thermodynamic fingerprints reveal an excellent correlation between gains in enthalpy and compensation by increased entropy costs. Moreover, 17 b exhibits a residence time in the range of a few seconds, clearly prolonged relative to residence times typically observed for carbohydrate-lectin interactions. Finally, initial tests regarding drug-like properties of 17 b demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range.

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Section: Isothermal Titration Calorimetry (Itc)supporting

confidence: 64%

From a Library of MAG Antagonists to Nanomolar CD22 Ligands

et al. 2011

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“…In addition, NMR studies have shown that the LG3 domain of agrin binds sialic acid in a Ca 2+ -dependent manner, whilst binding the glycosaminoglycans heparin and heparan sulfate bind independently of Ca 2+ . It remains unclear whether these observations may be relevant for α-dystroglycan binding to agrin (Sallum et al, 2007).…”

Section: Structural Analysis and Modeling Of Different α-Dg Binding Lmentioning

confidence: 99%

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains

Dempsey

Bigotti

Adams

et al. 2019

Front. Mol. Biosci.

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Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular α-DG associated noncovalently with a transmembrane β-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (α-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the α-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind α-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for α-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic α-DG non-binders such as laminin-α3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of α-DG, although it appears that this alone is insufficient to mediate significant α-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with α-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

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“…Precipitates were removed by sedimentation. Pulse-field gradient diffusion experiments (Sallum et al 2007;Whitehead et al 2022) calibrated against the internal standard DSS, gave an Rh of 9.1 ± 0.6 Å for a Zn 2+ -bound 2.4 mM WT sample at 25 o C, pH 6.2, 600 MHz, consistent with a monomeric oligomerization state for the Z7 domain under NMR conditions (Rua et al 2023;Whitehead et al 2022).…”

Section: Nmr Spectroscopymentioning

confidence: 84%

“…Precipitates were removed by sedimentation. Pulse-field gradient diffusion experiments (Sallum et al . 2007; Whitehead et al .…”

Section: Methodsmentioning

confidence: 99%

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

Rua,

Alexandrescu

2024

Preprint

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Domain Z7 of nuclear transcription factor ZNF711 has the consensus last metal-ligand H23 found in odd-numbered zinc-fingers of this protein replaced by a phenylalanine. Ever since the discovery of ZNF711 it has been thought that Z7 is probably non-functional because of the H23F substitution. The presence of H26 three positions downstream prompted us to examine if this histidine could substitute as the last metal ligand. The Z7 domain adopts a stable tertiary structure upon metal binding. The NMR structure of Zn2+-bound Z7 shows the classical ββα-fold of CCHH zinc fingers. Mutagenesis and pH titration experiments indicate that H26 is not involved in metal binding and that Z7 has a tridentate metal-binding site comprised of only residues C3, C6, and H19. By contrast, an F23H mutation that introduces a histidine in the consensus position forms a tetradentate ligand. The structure of the WT Z7 is stable causing restricted ring-flipping of phenyalanines 10 and 23. Dynamics are increased with either the H26A or F23H substitutions and aromatic ring rotation is no longer hindered in the two mutants. The mutations have only small effects on the Kd values for Zn2+and Co2+and retain the high thermal stability of the WT domain above 80oC. Like two previously reported designed zinc fingers with the last ligand replaced by water, the WT Z7 domain is catalytically active, hydrolyzing 4-nitophenyl acetate. We discuss the implications of naturally occurring tridentate zinc fingers for cancer mutations and drug targeting of notoriously undruggable transcription factors. Our findings that Z7 can fold with only a subset of three metal ligands suggests the recent view that most everything about protein structure can be predicted through homology modeling might be premature for at least the resilient and versatile zinc-finger motif.

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Thermodynamic and Structural Studies of Carbohydrate Binding by the Agrin-G3 Domain

Cited by 8 publications

References 63 publications

From a Library of MAG Antagonists to Nanomolar CD22 Ligands

From a Library of MAG Antagonists to Nanomolar CD22 Ligands

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains

Formerly degenerate seventh zinc finger domain from transcription factor ZNF711 rehabilitated by experimental NMR structure

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