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.

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti-tau antibody AT8.

Microtubule-associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF-tau). AT8 is a PHF-tau-specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30-fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF-tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202-209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR-L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody.

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.

An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations.

The Fc variant of IgG2, designated as IgG2σ, was engineered with V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement protein and consequently, immune effector functions. IgG2σ was compared to other previously well-characterized Fc 'muted' variants, including aglycosylated IgG1, IgG2m4 (H268Q/V309L/A330S/P331S, changes to IgG4), and IgG4 ProAlaAla (S228P/L234A/L235A) in its capacity to bind FcγRs and activate various immune-stimulatory responses. In contrast to the previously characterized muted Fc variants, which retain selective FcγR binding and effector functions, IgG2σ shows no detectable binding to the Fcγ receptors in affinity and avidity measurements, nor any detectable antibody-dependent cytotoxicity, phagocytosis, complement activity, or Fc-mediated cytokine release. Moreover, IgG2σ shows minimal immunogenic potential by T-cell epitope analysis. The circulating half-life of IgG2σ in monkeys is extended relative to IgG1 and IgG2, in spite of similar in vitro binding to recombinant FcRn. The three-dimensional structure of the Fc, needed for assessing the basis for the absence of effector function, was compared with that of IgG2 revealing a number of conformational differences near the hinge region of the CH2 domain that result from the amino acid substitutions. Modeling reveals that at least one of the key interactions with FcγRs is disrupted by a conformational change that reorients P329 to a position that prevents it from interacting with conserved W90 and W113 residues of the FcγRs. Inspection of the structure also indicated significant changes to the conformations of D270 and P329 in the CH2 domain that could negatively impact C1q binding. Thus, structural perturbations of the Fc provide a rationale for the loss of function. In toto, these properties of IgG2σ suggest that it is a superior alternative to previously described IgG variants of minimal effector function, for future therapeutic applications of non-immunostimulatory mAb and Fc-fusion platforms.

Canonical structures of short CDR-L3 in antibodies.

Despite sequence diversity, five out of six hypervariable loops in antibodies assume a limited number of conformations called canonical structures. Their correct identification is essential for successful prediction of antibody structure. This in turn requires regular updates of the classification of canonical structures to match the expanding experimental database. Antibodies with the eight-residue CDR-L3 represent the second most common type of antibodies after those with the nine-residue CDR-L3. We have analyzed all crystal structures of Fab and Fv with the eight-residue CDR-L3 and identified three major canonical structures covering 82% of a nonredundant set. In most cases, the canonical structure is defined by the absence or presence and position of a proline residue within the CDR.

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.

Structure and specificity of an antibody targeting a proteolytically cleaved IgG hinge.

The functional role of human antihinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656-1667. © 2014 Wiley Periodicals, Inc.

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.

Second antibody modeling assessment (AMA-II).

To assess the state of the art in antibody 3D modeling, 11 unpublished high-resolution x-ray Fab crystal structures from diverse species and covering a wide range of antigen-binding site conformations were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. The participants included: Accerlys Inc, Chemical Computer Group (CCG), Schrodinger, Jeff Gray's lab at John Hopkins University, Macromoltek, Astellas Pharma/Osaka University and Prediction of ImmunoGlobulin Structure (PIGS). The sequences of benchmark structures were submitted to the modelers and PIGS, and a set of models were generated for each structure. We provide here an overview of the organization, participants and main results of this second antibody modeling assessment (AMA-II). Also, we compare the results with the first antibody assessment published in this journal (Almagro et al., 2011;79:3050).

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.

Antibody modeling assessment.

A blinded study to assess the state of the art in three-dimensional structure modeling of the variable region (Fv) of antibodies was conducted. Nine unpublished high-resolution x-ray Fab crystal structures covering a wide range of antigen-binding site conformations were used as benchmark to compare Fv models generated by four structure prediction methodologies. The methodologies included two homology modeling strategies independently developed by CCG (Chemical Computer Group) and Accerlys Inc, and two fully automated antibody modeling servers: PIGS (Prediction of ImmunoGlobulin Structure), based on the canonical structure model, and Rosetta Antibody Modeling, based on homology modeling and Rosetta structure prediction methodology. The benchmark structure sequences were submitted to Accelrys and CCG and a set of models for each of the nine antibody structures were generated. PIGS and Rosetta models were obtained using the default parameters of the servers. In most cases, we found good agreement between the models and x-ray structures. The average rmsd (root mean square deviation) values calculated over the backbone atoms between the models and structures were fairly consistent, around 1.2 Å. Average rmsd values of the framework and hypervariable loops with canonical structures (L1, L2, L3, H1, and H2) were close to 1.0 Å. H3 prediction yielded rmsd values around 3.0 Å for most of the models. Quality assessment of the models and the relative strengths and weaknesses of the methods are discussed. We hope this initiative will serve as a model of scientific partnership and look forward to future antibody modeling assessments.

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.

FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD-specific STAT5 activation.

Myeloid cell leukemia-1 (MCL-1) is an essential survival factor for hematopoiesis. In humans, hematopoietic stem cells (HSCs) express MCL-1 at the highest level in response to FMS-like tyrosine kinase-3 (FLT3) signaling. We here show that this FLT3-dependent stem cell maintenance system also plays a critical role in survival of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). The CD34(+)CD38(-) LSC fraction expresses high levels of FLT3 as well as MCL-1, even compared with normal HSCs. Treatment with FLT3 ligand induced further MCL-1 up-regulation in LSCs in all AML cases tested. Interestingly, the group of samples expressing the highest levels of MCL-1 constituted AML with FLT3-internal tandem duplications (ITD). In FLT3-ITD AML cell lines, cells expressed a high level of MCL-1, and an inhibition of MCL-1 induced their apoptotic cell death. A tyrosine kinase inhibitor suppressed MCL-1 expression, and induced apoptosis that was reversed by the enforced MCL-1 expression. Finally, transduction of FLT3-ITD into HSCs strongly activated MCL-1 expression through its signal transducer and activator of transcription 5 (STAT5)-docking domains. This effect was completely abrogated when STAT5 activation was blocked. Thus, the acquisition of FLT3-ITD ensures LSC survival by up-regulating MCL-1 via constitutive STAT5 activation that is independent of wild-type FLT3 signaling.

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.

Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells.

An attractive target for therapeutic intervention is constitutively activated, mutant FLT3, which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing, and which is currently in late-stage clinical trials. However, the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel, structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here, we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487, which selectively targets mutant FLT3 protein kinase activity, is also shown to override PKC412 resistance in vitro, and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally, the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus, we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target, and which could potentially be used to override drug resistance in AML.

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.