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. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.

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. The structure revealed the binding epitope, explained the lack of cross-reactivity toward non-human species, and suggested the key-and-lock mechanism of antigen recognition. Most unexpectedly, the structure revealed a unique molecular topology of CD19. Rather than a tandem of c-type immunoglobulin folds predicted from the amino acid sequence, the extracellular domain of CD19 exhibits an elongated ß-sandwich formed by two immunoglobulin folds by swapping their C-terminal halves. This is the first structure of CD19, which has no sequence homologs.

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. The structure of the DARPin remains virtually unchanged. The conformational changes in IL-4 include a reorientation of the C-helix Trp91 side chain and repositioning of CD-loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL-4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical "hotspot." The structures also reveal how the DARPin neutralizes IL-4 signaling. Comparing the IL-4:DARPin complex structure with the structures of IL-4 bound to its receptors (Hage et al., Cell 1999; 97, 271-281; La Porte et al., Cell 2008, 132, 259-272), it is found that the DARPin binds to the same IL-4 face that interacts with the junction of the D1 and D2 domains of the IL-4Rα receptors. Signaling is blocked since IL-4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL-13α1 or the γc receptor.

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.

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. In the second round, CDR-H3 modeling was performed in the context of the correct environment provided by the crystal structures with CDR-H3 removed. In this report we describe the reference structures and present our assessment of the models. Some of the essential sources of errors in the predictions were traced to the selection of the structure template, both in terms of the CDR canonical structures and VL/VH packing. On top of this, the errors present in the Protein Data Bank structures were sometimes propagated in the current models, which emphasized the need for the curated structural database devoid of errors. Modeling non-canonical structures, including CDR-H3, remains the biggest challenge for antibody structure prediction.

C-terminal ß-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 ß-strand resulting in the formation of Tencon dimers in which the target-binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the ß-strand-swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non-glycine residue at position 77 and insertions of <4 residues may destabilize the ß-turn in the FG loop promoting ß-strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect ß-bridges in the FG regions that link the swapped dimers. The Tencon ß-swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain-swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties.

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

The crystal structure of an N-terminal ß-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. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N-terminal ß-strand swapping, leading to the formation of a stable compact dimer.

Crystal structure of D-serine dehydratase from Escherichia coli.

D-Serine dehydratase from Escherichia coli is a member of the ß-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. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the ß-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.

His-tag binding by antibody C706 mimics ß-amyloid recognition.

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of ß-amyloid (Aß) plaques. Aggregation of the Aß(42) peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti-Aß monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aß peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2(1) and C2. Although the Fab was crystallized in the presence of Aß(16), no peptide was observed in the crystals. The antigen-binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly-His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4-His5) stack against tryptophan residues in the central pocket of the antigen-binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his-tag binding by C706 resembles the Aß recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B-cell epitope of Aß. By similarity, residues Phe-Arg-His of Aß would be a major portion of the C706 epitope.

Expression, refolding and purification of a human interleukin-17A variant.

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.

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. This comparison indicates an induced-fit mechanism of antigen recognition through rigid-body rotation of the V(L) and V(H) domains. The magnitude of this rearrangement is one of the largest observed for antibody-protein interactions.

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. Fine purification entailed the application of a very shallow gradient in anion-exchange chromatography, resulting in the resolution of two separate complex peaks which had different crystallizabilities. Subsequent structure determination defined the epitopes of the respective antibodies and revealed a mechanistic hypothesis that is currently under investigation. The results also showed that cocrystallization with multiple noncompeting Fab fragments can be a viable path when an antigen complex with a single Fab proves to be recalcitrant to crystallization.

Promoting crystallization of antibody-antigen complexes via microseed matrix screening.

The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate.

Noncanonical conformation of CDR L1 in the anti-IL-23 antibody CNTO4088.

CNTO4088 is a monoclonal antibody to human IL-23. The X-ray structure of the Fab fragment revealed an unusual noncanonical conformation of CDR L1. Most antibodies with the kappa light chain exhibit a canonical structure for CDR L1 in which residue 29 anchors the CDR loop to the framework. Analysis of the residues believed to define the conformation of CDR L1 did not explain why it should not adopt a canonical conformation in this antibody. This makes CNTO4088 a benchmark case for developing prediction methods and structure-modeling tools.

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells.

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

Inexpensive robotic system for standard and fluorescent imaging of protein crystals.

Protein-crystallization imaging and classification is a labor-intensive process typically performed either by humans or by instruments that currently cost well over $100 000. This cost puts the use of crystallization-trial imaging outside the reach of most academic laboratories, and also start-up biotechnology firms, where resources are scarce. An imaging system has been designed and prototyped which automatically captures images from multi-well protein-crystallization experiments using both standard and fluorescent imaging techniques at a cost 28 times lower than current market rates. The machine uses a Panowin F1 3D printer as a base and controls it using G-code commands sent from a Python script running on a desktop computer. A graphical user interface (GUI) was developed to enable users to control the machine and facilitate image capture, classification and editing. A 488 nm laser diode and a 525 nm filter were incorporated to allow in situ fluorescent imaging of proteins trace-labeled with a fluorophore, Alexa Fluor 488. The instrument was primarily designed using a 3D printer and augmented using commercially available parts, and this publication aims to serve as a guide for comparable in-laboratory robotics projects.

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. To gain insight into the structural determinants of antigen recognition, the crystal structure of M116 Fab was determined in complex with CCL17 and in the unbound form. Comparison of the structures revealed an unusual induced-fit mechanism of antigen recognition that involves cis-trans isomerization in two CDRs. The structure of the CCL17-M116 complex revealed the antibody binding epitope, which does not overlap with the putative receptor epitope, suggesting that the current model of chemokine-receptor interactions, as observed in the CXCR4-vMIP-II system, may not be universal.

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. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.

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 and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

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

BACKGROUND: ß-Amyloid (Aß) peptide is believed to play a pivotal role in the development of Alzheimer's disease. Passive immunization with anti-Aß monoclonal antibodies may facilitate the clearance of Aß in the brain and may thus prevent the downstream pathology. Antibodies targeting the immunodominant N-terminal epitope of Aß and capable of binding both the plaques and soluble species have been most efficacious in animal models. Structural studies of such antibodies with bound Aß peptides provided the basis for understanding the mechanisms of action and the differences in potency. To gain further insight into the structural determinants of antigen recognition and the preferential Aß conformations, we determined the crystal structure of murine antibody C706 in complex with the N-terminal Aß 1-16 peptide sequence. METHODS: The antigen-binding fragment of C706 was expressed in HEK293 cells and was crystallized in complex with the Aß peptide. The X-ray structure was determined at 1.9-Å resolution. RESULTS: The binding epitope of C706 is centered on residues Arg5 and His6, which provide the majority of interactions. Unlike most antibodies, C706 recognizes a coiled rather than extended conformation of Aß. CONCLUSIONS: Comparison with other antibodies targeting the N-terminal section of Aß suggests that the conformation of the bound peptide may be linked to the immunization protocol and may reflect the preference for the extended conformation in the context of a longer Aß peptide as opposed to the coiled conformation in the isolated short peptide.