The structure of the anti‐c‐myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops

Krauß, Norbert; Wessner, Helga; Welfle, Karin; Welfle, Heinz; Scholz, C.; Seifert, Martina; Zubow, Kristina; Aÿ, Jacqueline; Hahn, Michael G.; Scheerer, Patrick; Skerra, Arne; Höhne, Wolfgang

doi:10.1002/prot.22080

Cited by 27 publications

(23 citation statements)

References 58 publications

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“…Among ∼1,200 structures of antibody-antigen complexes in the Protein Data Bank, we found a single example, anti-C-myc antibody 9E10, that seems to exhibit antigen clasping. However, the binding mode of 9E10 conforms to the convention of anti-peptide antibodies because one Fab dominates peptide recognition by capturing the peptide into a deep cleft created with long CDRs, and this antibody showed no cooperative binding (23). By contrast, in our antibodies, two Fab molecules synergistically contribute to peptide recognition.…”

Section: Discussionmentioning

confidence: 62%

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

Hattori

Lai

Dementieva

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.antibody engineering | epigenetics | antibody validation | protein-protein interaction | data reproducibility

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Section: Discussionmentioning

confidence: 62%

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

Hattori

Lai

Dementieva

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Whereas we attached DNA handles to both ends of A2, holding A2 approximately 260 nm away from each bead and preventing nonspecific interactions, the other study attached DNA at only one end of the A2 construct. Their A2 N terminus contained a ðc-mycÞ 4 tag that directly bound an anti-c-myc antibody-coated bead, placing A2 approximately 840 nm away from the DNAbound bead but only 0-30 nm away from the ðc-mycÞ 4 tag-bound bead [depending on whether the antibody coupled through its Fab or Fc region to the bead, which of the four c-myc tags the antibody (20) bound to, and fluctuations in the flexible c-myc tags and antibody at low force]. DNA handles are not only much longer but also much stiffer than the c-myc tags.…”

Section: Discussionmentioning

confidence: 99%

Calcium stabilizes the von Willebrand factor A2 domain by promoting refolding

Springer

2012

Proc. Natl. Acad. Sci. U.S.A.

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Von Willebrand factor (VWF) is a large, multimeric plasma glycoprotein that critically mediates hemostasis at sites of vascular injury. Very large VWF multimers have the greatest thrombogenic activity, which is attenuated by cleavage in the A2 domain by the metalloproteinase ADAMTS13. ADAMTS13 proteolysis requires mechanical force to expose the scissile bond and is regulated by a calcium-binding site within A2. In this study, we characterized the interaction between VWF A2 and calcium by examining the effect of calcium on VWF A2 stability and mechanical unfolding and refolding. Isothermal calorimetry yielded a calcium binding K d ¼ 3.8 AE 1.0 μM and reversible thermal denaturation showed that 5 mM calcium stabilized the unfolding transition from 56.7 AE 0.1 to 69.1 AE 0.1°C. Using optical tweezers to apply tensile force to single domains, we found that calcium did not affect VWF A2 unfolding, but rather enhanced refolding kinetics fivefold, resulting in a 0.9 kcal∕mol stabilization in the folding activation energy in the presence of calcium. Taken together, our data demonstrate that VWF binds calcium at physiologic calcium concentrations and that calcium stabilizes VWF A2 by accelerating refolding. V on Willebrand factor (VWF) is a large plasma glycoprotein that critically mediates hemostasis at sites of vascular injury. Secreted by endothelial cells and platelets in response to thrombogenic stimuli, VWF circulates as ultralong, disulfide-bonded concatamers (1) reaching >12 MDa and >15 μm in size (2, 3). Each VWF monomer is comprised of multiple domains, including domains that bind clotting factor VIII, collagen, platelets, and integrins (1). The A2 domain contains a cleavage site recognized by the enzyme ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), which regulates VWF length. The largest VWF concatamers mediate the formation of a platelet plug through multivalent binding to collagen and platelet membrane glycoprotein Ib. Mutations within VWF cause von Willebrand's disease (VWD), a heterogeneous set of bleeding disorders that underscore VWF function in normal physiology (4).VWF function is critically determined by concatamer length. Healthy individuals exhibit a heterogeneous distribution of VWF concatamers, whose size is regulated through specific cleavage by ADAMTS13. Absence of ADAMTS13 function causes thrombocytic thrombocytopenic purpura (TTP), a life-threatening condition characterized by extensive microvascular thromboses that result in tissue ischemia and infarction. Neurologic symptoms and renal failure are often described, and the condition resolves upon replenishment of ADAMTS13 with plasma exchange (5). In contrast, genetic mutations that shift the VWF size distribution to smaller multimers, comprising type 2 VWD, present with increased bleeding due to reduced VWF thrombogenicity (6). Subtype 2A VWD mutations cluster within the A2 domain, which bears the ADAMTS13 cleavage site, and whose structure illuminates mechanisms that regulate VWF cleavage (7).Flu...

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“…1a). This level of affinity is 3–4 orders of magnitude greater than those for FLAG/antibody and c-myc/antibody (both ~400 nM K d ) (9,10) and His 6 -tag/immobilized metal (~10 µM) (11). The tag can be readily cleaved with thrombin as expected (Supplementary Fig.…”

mentioning

confidence: 84%

A Peptide Tag System for Facile Purification and Single-Molecule Immobilization

et al. 2009

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A peptide fusion tag and accompanying recombinant capture reagents have been developed based on the peptide-PDZ domain interaction and affinity clamps, a new class of affinity reagent. This system allows for single-step purification under mild conditions and stable capture of a tagged protein. The sub-nanomolar affinity, high force resistance (>30 pN), small size (~25kDa, ~1/6 of IgG) and monomeric nature of the affinity clamp are all superior features for many applications, in particular single-molecule measurements.Genetic fusion tags and accompanying capture reagents facilitate detection, purification and immobilization of recombinant proteins, and thus they have become an integral component in biochemical, biophysical and cellular investigations (1). Short peptide tags, often called epitope tags, are minimally invasive and useful for detection and purification. Folded protein tags often serve additional roles in increasing the solubility and expression yields. Together, available tag systems satisfy the requirements for standard applications in protein expression, detection and purification.Single-molecule measurements are uniquely powerful in their ability to reveal the molecular mechanisms underlying motility, conformational changes and force generation. Fusion tags are routinely used for immobilization and detection in single-molecule measurements but this type of research poses demanding requirements for fusion tags and their accompanying capture reagents. (i) A fusion tag should be short so as to minimally affect the structure and oligomerization state of the protein of interest. (ii) The interaction between a tag and its capture reagent must be of high affinity so that it is stably formed even at very low protein concentrations (typically ~10 pM). (iii) A capture reagent should be monomeric and highly specific. (iv) Multiple, mutually orthogonal tag/capture reagent systems should be available. These requirements render a majority of commonly used tag/capture reagent systems unsuitable for single-molecule measurements.The current standard is to combine the biotinylation tag/(strept)avidin system with a monoclonal antibody (mAb) system (2-5). Although the biotinylation tag/(strept)avidin system is widely used due to its high affinity and specificity(6), (strept)avidin is a tetrameric protein that can potentially bring multiple molecules to close proximity (Fig. 1a). A GFP/anti-GFP mAb system offers some unique advantages, but the complex is large and can also force artificial dimerization, making it a suboptimal solution (Fig. 1a). Furthermore, we have experienced batch-to-batch inconsistency of monoclonal antibodies. Recent commercial *To whom correspondence should be addressed. Phone: (773)702-5073 (SK) or (773) preparations of anti-GFP antibodies contained actin-binding contaminants that interfered with our work on myosin motors. We therefore sought an alternative that would combine high affinity with high specificity, and would also complement the streptavidin-biotin links that we use elsewhe...

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The structure of the anti‐c‐myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops

Cited by 27 publications

References 58 publications

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

Calcium stabilizes the von Willebrand factor A2 domain by promoting refolding

A Peptide Tag System for Facile Purification and Single-Molecule Immobilization

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