Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium.

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet.

Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector.

Crystal structure of B-cell co-receptor CD19 in complex with antibody B43 reveals an unexpected fold.

CD19 is a transmembrane protein expressed on malignant B cells, but not in other lineages or other tissues, which makes it an attractive target for monoclonal antibody-mediated immunotherapy. Anti-CD19 antibody B43 was utilized in a bispecific T-cell engager (BiTE) blinatumomab that demonstrated potency for the treatment of relapsed acute lymphoblastic leukemia. To gain insight into the mechanism of action of the antibody, the crystal structure of B43 Fab was determined in complex with CD19 and in the unbound form.

Structural insights into humanization of anti-tissue factor antibody 10H10.

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries.

Structural insights into chemokine CCL17 recognition by antibody M116.

The homeostatic chemokine CCL17, also known as thymus and activation regulated chemokine (TARC), has been associated with various diseases such as asthma, idiopathic pulmonary fibrosis, atopic dermatitis and ulcerative colitis. Neutralization of CCL17 by antibody treatment ameliorates the impact of disease by blocking influx of T cells. Monoclonal antibody M116 derived from a combinatorial library shows potency in neutralizing CCL17-induced signaling.

A coiled conformation of amyloid-β recognized by antibody C706.

Background: β-Amyloid (Aβ) peptide is believed to play a pivotal role in the development of Alzheimer's disease. Passive immunization with anti-Aβ monoclonal antibodies may facilitate the clearance of Aβ in the brain and may thus prevent the downstream pathology. Antibodies targeting the immunodominant N-terminal epitope of Aβ and capable of binding both the plaques and soluble species have been most efficacious in animal models.

Crystal structure of tissue factor in complex with antibody 10H10 reveals the signaling epitope.

Tissue factor (TF) initiates the extrinsic pathway of blood coagulation through sequential binding and activation of coagulation factors VII (FVII) and X (FX). In addition, through activation of G-protein-coupled protease activated receptors (PARs) TF induces cell signaling that is related to cancer, angiogenesis and inflammation. Monoclonal antibodies (mAbs) proved to be a useful tool for studying the interplay between TF signaling and coagulation.

Crystal structure of CD27 in complex with a neutralizing noncompeting antibody.

CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70.

Epitope-dependent mechanisms of CD27 neutralization revealed by X-ray crystallography.

CD27 is a T and B cell co-stimulatory protein of the TNF receptor superfamily dependent on the availability of the TNF-like ligand CD70. Two anti-CD27 neutralizing monoclonal antibodies were obtained from mouse hybridoma and subsequently humanized and optimized for binding the target. The two antibodies are similar in terms of their CD27-binding affinity and ability to block NF-κB signaling, however their clearance rates in monkeys are very different.

Conformational flexibility of an anti-IL-13 DARPin†.

Designed ankyrin repeat proteins (DARPin) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9 in the unbound form and in complex with IL-13 were determined at high resolution.

Structural diversity in a human antibody germline library.

To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions.

Induced conformational change in human IL-4 upon binding of a signal-neutralizing DARPin.

The crystal structure of DARPin 44C12V5 that neutralizes IL-4 signaling has been determined alone and bound to human IL-4. A significant conformational change occurs in the IL-4 upon DARPin binding. The DARPin binds to the face of IL-4 formed by the A and C α-helices.

Protein crystallization with microseed matrix screening: application to human germline antibody Fabs.

The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs.

Antibody modeling assessment II. Structures and models.

To assess the state-of-the-art in antibody structure modeling, a blinded study was conducted. Eleven unpublished Fab crystal structures were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. In the first round, each participant submitted three non-ranked complete Fv models for each target.

Structural evidence for a constrained conformation of short CDR-L3 in antibodies.

Three Fab structures used as targets in the Antibody Modeling Assessment presented a challenge for modeling CDR-L3 due to deviations from the most typical canonical structure. In all three antibodies CDR-L3 has eight residues, which is one residue shorter than usual, and has a conformation that is rarely observed in crystal structures. We analyzed the sequence and structural determinants of this conformation and found that the "short" CDR-L3 is remarkably rigid and retains the conformation in the interactions with antigens and neighboring CDRs.

N-terminal β-strand swapping in a consensus-derived alternative scaffold driven by stabilizing hydrophobic interactions.

The crystal structure of an N-terminal β-strand-swapped consensus-derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain.

C-terminal β-strand swapping in a consensus-derived fibronectin Type III scaffold.

The crystal structures of six different fibronectin Type III consensus-derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C-terminal β-strand resulting in the formation of Tencon dimers in which the target-binding surface is blocked.

IgG2 Fc structure and the dynamic features of the IgG CH2-CH3 interface.

The analyses of two human IgG2 Fc structures, determined in different crystal forms, and the comparison with IgG1 Fc structures reveals molecular features that are involved in accommodating and stabilizing structural conformations. In the IgG2 Fc structures relative positions of the CH2 domains with respect to the CH3 domains vary significantly from those observed for IgG1 Fc structures in similar unit cells. The analysis reveals that the movement of the CH2 domain in all of the Fc structures results from a pivoting around a highly conserved ball-and-socket-like joint in which the CH2 L251 side chain (the ball) interacts with a pocket (the socket) formed by CH3 M428, H429, E430, and H435.

Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure.

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation.

Structural basis for high selectivity of anti-CCL2 neutralizing antibody CNTO 888.

Human CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), is a member of the β chemokine family whose actions are mediated through the G-protein-coupled receptor CCR2. Binding of CCL2 to its receptor CCR2 triggers calcium mobilization and chemotaxis. CCL2 is implicated in the pathogenesis of certain inflammatory diseases and cancer.

Design of novel FN3 domains with high stability by a consensus sequence approach.

The use of consensus design to produce stable proteins has been applied to numerous structures and classes of proteins. Here, we describe the engineering of novel FN3 domains from two different proteins, namely human fibronectin and human tenascin-C, as potential alternative scaffold biotherapeutics. The resulting FN3 domains were found to be robustly expressed in Escherichia coli, soluble and highly stable, with melting temperatures of 89 and 78°C, respectively.

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement.

Crystallization of a challenging antigen-antibody complex: TLR3 ECD with three noncompeting Fabs.

The mechanism of action of therapeutic antibodies can be elucidated from the three-dimensional crystal structures of their complexes with antigens, but crystallization remains the primary bottleneck to structure determination. Methods that resulted in the successful crystallization of TLR3 ECD in complex with Fab fragments from three noncompeting, neutralizing anti-TLR3 antibodies are presented. The crystallization of this 238 kDa complex was achieved through fine purification of the quaternary complex of TLR3 with the three Fab fragments combined with microseed matrix screening and additive screening.

Antigen recognition by antibody C836 through adjustment of V(L)/V(H) packing.

C836 is a neutralizing monoclonal antibody to human interleukin IL-13 generated by mouse immunization. The crystal structure of the C836 Fab was determined at 2.5 Å resolution and compared with the IL-13-bound form determined previously.