Jonathan M. Wojciak

The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen

The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant roles in angiogenesis, heart disease, and cancer. LT1009 (also known as sonepcizumab) is a humanized monoclonal antibody that binds S1P with high affinity and specificity. Because the antibody is currently in clinical trials, it is important to confirm by structural and biochemical analyses that it binds its target in a predictable manner. Therefore, we determined the structure of a complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 Å resolution. The antibody employs unique and diverse strategies to recognize its antigen. Two metal ions bridge complementarity determining regions from the antibody light chain and S1P. The coordination geometry, inductively coupled plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical assays suggest that these are Ca2+. The amino alcohol head group of the sphingosine backbone is recognized through hydrogen bonding interactions from 1 aa side chain and polypeptide backbone atoms of the antibody light and heavy chains. The S1P hydrophobic tail is almost completely enclosed within a hydrophobic channel formed primarily by the heavy chain. Both treatment of the complex with metal chelators and mutation of amino acids in the light chain that coordinate the metal atoms or directly contact the polar head group abrogate binding, while mutations within the hydrophobic cavity also decrease S1P binding affinity. The structure suggests mechanistic details for recognition of a signaling lipid by a therapeutic antibody candidate. Moreover, this study provides direct structural evidence that antibodies are capable of using metals to bridge antigen:antibody complexes.

Arm-site binding by λ-integrase: Solution structure and functional characterization of its amino-terminal domain

The integrase protein (Int) from bacteriophage λ catalyzes the insertion and excision of the viral genome into and out of Escherichia coli. It is a member of the λ-Int family of site-specific recombinases that catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria, and yeast and belongs to the subset of this family that possesses two autonomous DNA-binding domains. The heterobivalent properties of Int can be decomposed into a carboxyl-terminal domain that executes the DNA cleavage and ligation reactions and a smaller amino-terminal domain that binds to an array of conserved DNA sites within the phage arms, thereby arranging Int protomers within the higher-order recombinogenic complex. We have determined that residues Met-1 to Leu-64 of Int constitute the minimal arm-type DNA-binding domain (INT-DBD1–64) and solved the solution structure by using NMR. We show that the INT-DBD1–64 is a novel member of the growing family of three-stranded β-sheet DNA-binding proteins, because it supplements this motif with a disordered amino-terminal basic tail that is important for arm-site binding. A model of the arm-DNA-binding domain recognizing its cognate DNA site is proposed on the basis of similarities with the analogous domain of Tn916 Int and is discussed in relation to other features of the protein.

Regulation of Directionality in Bacteriophage λ Site-specific Recombination: Structure of the Xis Protein

Abstract Upon induction of a bacteriophage λ lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual “winged”-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single α-helix and loop structure (“wing”), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase–Xis–DNA complex required for phage excision.

NMR structure of the Tn916 integrase-DNA complex.

The integrase protein catalyzes the excision and integration of the Tn 916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel β-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-PpoI–DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent ˜35° and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.

Site-specific DNA binding using a variation of the double stranded RNA binding motif

The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded β-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.

Finding the function buried in SAND.

The SAND domain is present in many proteins that have been linked to chromatin-dependent transcriptional regulation and human disease, but their function has remained unknown. Recent studies have revealed that these modules are DNA binding domains of novel molecular structure.

Identification of the λ integrase surface that interacts with Xis reveals a residue that is also critical for Int dimer formation

Lambda integrase (Int) is a heterobivalent DNA-binding protein that together with the accessory DNA-bending proteins IHF, Fis, and Xis, forms the higher-order protein–DNA complexes that execute integrative and excisive recombination at specific loci on the chromosomes of phage λ and its Escherichia coli host. The large carboxyl-terminal domain of Int is responsible for binding to core-type DNA sites and catalysis of DNA cleavage and ligation reactions. The small amino-terminal domain (residues 1–70), which specifies binding to arm-type DNA sites distant from the regions of strand exchange, consists of a three-stranded β-sheet, proposed to recognize the cognate DNA site, and an α-helix. We report here that a site on this α-helix is critical for both homomeric interactions between Int protomers and heteromeric interactions with Xis. The mutant E47A, which was identified by alanine-scanning mutagenesis, abolishes interactions between Int and Xis bound at adjacent binding sites and reduces interactions between Int protomers bound at adjacent arm-type sites. Concomitantly, this residue is essential for excisive recombination and contributes to the efficiency of the integrative reaction. NMR titration data with a peptide corresponding to Xis residues 57–69 strongly suggest that the carboxyl-terminal tail of Xis and the α-helix of the aminoterminal domain of Int comprise the primary interaction surface for these two proteins. The use of a common site on λ Int for both homotypic and heterotypic interactions fits well with the complex regulatory patterns associated with this site-specific recombination reaction.

Biochemical and structural characterization of lysophosphatidic Acid binding by a humanized monoclonal antibody.

Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the X-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 Å resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 Å, respectively. The variable CDR (complementarity-determining region) loops at the antigen binding site adopt nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy- and light-chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent-exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically on the basis of the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling.

NMR structure of the Tn 916 integrase–DNA complex

The integrase protein catalyzes the excision and integration of the Tn 916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel β-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-PpoI–DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent ˜35° and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.