The de Novo Design of an Antibody Combining Site

Essen, Lars‐Oliver; Skerra, Arne

doi:10.1006/jmbi.1994.1284

Cited by 42 publications

(15 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…deviation was 0.66A between the D1 domains determined in two crystal forms (Ryu, Truneh, Sweet & Hendrickson, 1994). The two V domains in protein Rei (Epp et al, 1975) and in protein War (Huang et al, 1994) and in protein M29b (Essen & Skerra, 1994) that were determined with higher, approximately 2,~,, resolution data are more similar to each other, the r.m.s, deviation is 0.35, 0.36 and 0.35 A, respectively. In general the cores of the domains with identical sequences became more similar with increased resolution of the diffraction data, and as the refinement progressed.…”

Section: Structure Determination: Molecular Replacementmentioning

confidence: 71%

Pitfalls of Molecular Replacement: the Structure Determination of an Immunoglobulin Light-Chain Dimer

Huang¹,

Ainsworth²,

Solomon³

et al. 1996

Acta Cryst D

View full text Add to dashboard Cite

The structure of protein Cle, a human light-chain dimer from the ,;AII subgroup, was determined using 2.6A data; the R value is 18.4%. The structure was solved, after a false start, by molecular replacement with the ;.II/V Mcg protein as a search structure. When the refinement did not proceed beyond an R value of 27 %, it was discovered that while the constant domains were in their correct positions in the unit cell, the incorrect variable domains were used for defining the molecule. The correct solution required a rotation of 180 around the local twofold axis that relates the two constant domains of the dimer. The correct variable domain positions overlap about 70% of the same volume as the incorrect ones of a symmetry-related molecule. The refinement distorted the geometries of the domains. Though the constant domains were in their correct positions, the r.m.s. (root-mean-square) deviation of the Co~ atom position was 1.2A when the two constant domains were compared. For the correct structure, this value is 0.5 A,. The q9 and ~p angles, the r.m.s, chiral value and the free R value, even when calculated a posteriori, were good indicators of the correctness of the structure. The quaternary structure of the Cle molecule is similar to that in Mcg (crystallized from ammonium sulfate); the elbow bend is 115. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 ,~, closer than in Mcg or variable dimer Rei. The hydrogen bonding at the interface of the two domains is novel. Residues Tyr36 from both monomers form a hydrogen bond that is part of a network with the Gin89 residues from both monomers. For the first time hydrogen bonds were observed between the main-chain peptide N and O atoms of the complementarity-determining region CDR2 and CDR3 segments of both monomers.

show abstract

Section: Structure Determination: Molecular Replacementmentioning

confidence: 71%

Pitfalls of Molecular Replacement: the Structure Determination of an Immunoglobulin Light-Chain Dimer

Huang¹,

Ainsworth²,

Solomon³

et al. 1996

Acta Cryst D

View full text Add to dashboard Cite

show abstract

“…There appears to be a wide range of interaction strengths and in one case there was little association at all [21], while in another the addition of isopropanol disrupted it extensively [22]. Hence, stabilization by scFv or dsFv approaches has been extensively pursued.…”

Section: Discussionmentioning

confidence: 99%

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

et al. 1995

View full text Add to dashboard Cite

A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the saltbridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60°C to 69°C. The ds-scFv form thus combines the stable monomeric form of the disulphide form wifh the expression advantages of the scFv.

show abstract

“…3 and S3, identified by their crystal structure PDB ID, are as follows: 2C1P, anti-finrozole (drug candidate); 75 3CFB, anti-trans-stilbene (blue fluorescent complex); 76 1FLR, anti-fluorescein (quenched fluorescent dye); 77 1LVE, LEN myeloma V L dimer; 78 1QAC, LEN Q89L mutant V L dimer; 24 2Q1E, amyloidogenic mutant V L dimer; 79 2RHE, Bence-Jones V L dimer; 80 1IVL, synthetic V L dimer. 81 …”

Section: Methodsmentioning

confidence: 99%

Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces

Szent-Györgyi

Stanfield

Andreko

et al. 2013

Journal of Molecular Biology

125

View full text Add to dashboard Cite

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents.

show abstract

The de Novo Design of an Antibody Combining Site

Cited by 42 publications

References 0 publications

Pitfalls of Molecular Replacement: the Structure Determination of an Immunoglobulin Light-Chain Dimer

Pitfalls of Molecular Replacement: the Structure Determination of an Immunoglobulin Light-Chain Dimer

Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces

Contact Info

Product

Resources

About