Antibody fragments as tools in crystallography

Griffin, Laura; Lawson, Alastair D. G.

doi:10.1111/j.1365-2249.2011.04427.x

Cited by 60 publications

(48 citation statements)

References 61 publications

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“…The adjunct provides additional, non-glycosylated polypeptide surface to the IR310.T complex and in so doing likely increases its propensity for crystallization. As such, it acts as a "crystallization chaperone" (16). In addition, attachment of the Fv was found to maintain solubility of IR310.T after endoglycosidase treatment and thus increase protein purification yield.…”

Section: Discussionmentioning

confidence: 99%

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Lawrence

Margetts

Menting

et al. 2016

Journal of Biological Chemistry

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Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucinerich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor ␣-chain (termed ␣CT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective ␣CT residues Phe 701 and Phe 705 . The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor.In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extracellular region and that had affinities in the high nanomolar to low micromolar range (1). Based on competition studies, the putative epitopes of these peptides were grouped into three non-overlapping sites, termed Sites 1, 2, and 3.3 Some Site 1 peptides were able to activate the receptor tyrosine kinase and act as agonists in an insulin-dependent fat cell assay, whereas Site 2 and Site 3 peptides were found to act as antagonists both in phosphorylation and fat cell assays. The highest affinity Site 1 peptides contained the motif FYXWF, whereas Site 2 peptides were characterized by either a short or long disulfide loop (1).Optimized versions of the Site 1 and Site 2 peptides were subsequently described (2), in particular those that were linked in tandem in various ways. Several of these linked peptides were found to function as either potent antagonists (e.g. peptide S661, a Site 1-Site 2 combination) or as potent agonists (e.g. peptide S597, a Site 2-Site 1 combination), depending on the order in which the individual Site 1 and Site 2 peptides were linked (2, 3). In earlier studies (4, 5), we provided isothermal titration calorimetry (ITC) 4 data that suggested how Site 1 peptides might bind to the insulin receptor. The insulin receptor (IR) itself is a disulfide-linked (␣␤) 2 homodimer; each ␣␤ monomer comprises, from its N terminus, two homologous leucine-rich repeat domains (L1 and L2) separated by a cysteine-rich region (CR) comprising eight disulfide-linked modules (6). These domains are followed by three fibronectin type III d...

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Section: Discussionmentioning

confidence: 99%

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Lawrence

Margetts

Menting

et al. 2016

Journal of Biological Chemistry

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“…We note that this mouse structure of IR ectodomain, comprising residues Pro31-Ser682 and mutations to stabilize the structure, was resolved in the presence of antibody fragments as crystallization chaperones to aid the structural determination of the IR ectodomain [15]. The antibody fragments serve in minimizing the conformational heterogeneity of IR through locking or clamping the IR ectodomain in a particular subset of conformations [54], whereas we performed the NMA in the absence of the antibody fragments. The integration of multiple methods is essential for obtaining a better picture of the IR mechanisms.…”

Section: Structural Data For Proposing Potential Therapeutic Strategiesmentioning

confidence: 99%

Harnessing Structural Data of Insulin and Insulin Receptor for Therapeutic Designs

et al. 2015

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To lower blood glucose concentration, insulin binds to insulin receptor (IR) that possesses two distinct insulin binding sites to trigger downstream signaling events leading to an increased uptake of glucose into muscle and fat cells. Comprehensive understandings of structural and dynamic mechanisms of insulin and its receptor are essential to design therapeutic agents for treating and delaying the onset of diabetes that affects over 347 million people worldwide. No full-length IR structure is available hitherto. Harnessing the currently available and state-of-the-art sequence and structural data, we have reviewed the insulin, IR, its extracellular domains and transmembrane domain, to derive structure-based clues to regulate aberrant insulin and its receptor. To propose testable hypotheses and future experiments, we have performed literature review, text mining, multiple structural clustering and normal mode analysis on insulin and its receptor. It appears that insulin-receptor interaction involves allostery and conformational changes (including rotation and tilting) to overcome steric clashes. To target a particular aberrant isoform of IRs, we need to identify the subtle yet distinct differences between IR isoforms. To improve the life quality of diabetics, better structure-based designs of insulin mimetics, formulation and nanotechnology-based delivery are required; efforts to bring them to patients necessitate thorough structural understandings of insulin and its receptor.

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“…The bound antibody fragment stabilizes the protein conformation and reduces structural heterogeneity, as well as provides additional crystal contacts. 3 The use of antibody tools to aid crystallization of "uncrystallizable" targets has been well characterized using both scFvs 78 and antigen-binding fragments (Fabs). 79 Alongside other protein affinity tools including DARPins, 80 work has also been carried out using nanobodies that are more stable than heterodimeric antibodies.…”

Section: Antibody Tools In Structural Studiesmentioning

confidence: 99%

“…3 The use of antibody tools to aid crystallization of "uncrystallizable" targets has been well characterized using both scFvs 78 and antigen-binding fragments (Fabs). 79 Alongside other protein affinity tools including DARPins, 80 work has also been carried out using nanobodies that are more stable than heterodimeric antibodies. 81 Wu et al 82 have described the successful use of the V H H scaffold (Fig.…”

Section: Antibody Tools In Structural Studiesmentioning

confidence: 99%

“…Once a lead small-molecule drug candidate has been identified, affinity reagents can be applied to aid crystallography of the target protein, providing structural and ligand interaction data to drive lead optimization. 3 Some specific aspects of the use of antibody tools have recently been described in more detail, including their use in the stabilization and sensing of specific protein conformations. 4 This review provides a broad commentary on the technical use of antibodies and other protein affinity reagents.…”

Section: Introductionmentioning

confidence: 99%

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The Use of Antibodies in Small-Molecule Drug Discovery

et al. 2014

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Antibodies are powerful research tools that can be used in many areas of biology to probe, measure, and perturb various biological structures. Successful drug discovery is dependent on the correct identification of a target implicated in disease, coupled with the successful selection, optimization, and development of a candidate drug. Because of their specific binding characteristics, with regard to specificity, affinity, and avidity, coupled with their amenability to protein engineering, antibodies have become a key tool in drug discovery, enabling the quantification, localization, and modulation of proteins of interest. This review summarizes the application of antibodies and other protein affinity reagents as specific research tools within the drug discovery process.

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Antibody fragments as tools in crystallography

Cited by 60 publications

References 61 publications

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Harnessing Structural Data of Insulin and Insulin Receptor for Therapeutic Designs

The Use of Antibodies in Small-Molecule Drug Discovery

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