Structural studies of blood coagulation factor VIII in complexes with inhibitory antibodies

Normal blood clotting is regulated by blood plasma protein factors in a blood coagulation cascade. Deficiency in one of these proteins, Factor VIII (FVIII), causes the disease hemophilia A, in which the patient is unable to form blood clots. Treatment of hemophilia A involves the infusion of FVIII, and the most common complication in this treatment is the development of antibodies against the therapeutic infusions. Two mouse monoclonal anti-human antibodies, 3E6 and G99, were used as models of inhibitory action against FVIII by binding to the C-terminal C2 domain. A classical antibody, 3E6, blocks the ability of FVIII to bind von Willebrand Factor, a circulatory partner. The classical antibody thereby reduces the stability of FVIII in plasma. A nonclassical antibody, G99, blocks the activation of FVIII, inhibiting the role of FVIII in the blood coagulation cascade. In order to study the inhibitory action of classical and nonclassical antibodies, 3 complexes were prepared, C2:3E6, C2:G99, and 3E6:C2:G99. Small angle X-ray scattering was used to study the structures of the C2 domain in complex with the antigen binding fragment (Fab) of each antibody and in complex with both Fabs simultaneously. Reproducible, low-resolution models of the complexes were generated from scattering data using a software suite from the European Molecular Biology Laboratory. The scattering data for 3E6:C2:G99 was used to generate an elongated shape with antibodies on opposite sides of the C2 domain. Extensive crystal trials were undertaken to prepare for future X-ray crystallography studies of the complexes

Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity

The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein-DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites

Characterization and Solution Structure of the Factor VIII C2 Domain in a Ternary Complex with Classical and Non-classical Inhibitor Antibodies

The most significant complication for patients with severe cases of congenital or acquired hemophilia A is the development of inhibitor antibodies against coagulation factor VIII (fVIII). The C2 domain of fVIII is a significant antigenic target of anti-fVIII antibodies. Here, we have utilized small angle x-ray scattering (SAXS) and biochemical techniques to characterize interactions between two different classes of anti-C2 domain inhibitor antibodies and the isolated C2 domain. Multiple assays indicated that antibodies 3E6 and G99 bind independently to the fVIII C2 domain and can form a stable ternary complex. SAXS-derived numerical estimates of dimensional parameters for all studied complexes agree with the proportions of the constituent proteins. Ab initio modeling of the SAXS data results in a long kinked structure of the ternary complex, showing an angle centered at the C2 domain of ∼130°. Guided by biochemical data, rigid body modeling of subunits into the molecular envelope of the ternary complex suggests that antibody 3E6 recognizes a C2 domain epitope consisting of the Arg2209–Ser2216 and Leu2178–Asp2187 loops. In contrast, antibody G99 recognizes the C2 domain primarily through the Pro2221–Trp2229 loop. These two epitopes are on opposing sides of the fVIII C2 domain, are consistent with the solvent accessibility in the context of the entire fVIII molecule, and provide further structural detail regarding the pathogenic immune response to fVIII

Evolutionary origins of C-terminal (GPP)n 3-hydroxyproline formation in vertebrate tendon collagen.

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species

Protein sequence alignment of the collagen (GPP)_n motif from phylogenetically diverse animals.

<p>Conservation of the (GPP)_n motif is shown in red for fibrillar collagens from early chordates through amphibians, birds and mammals. Genomic sequences are from Ensembl. Lamprey and ciona collagen sequences are from <i>Petromyzon marinus</i> transcript: COL2A1 ENSPMAT00000009617 and <i>Ciona intestinalis</i> transcript: FCOL1 ENSCINT00000014311, respectively.</p

Mass spectra of (GPP)_n containing tryptic peptides from adult animal tendons.

<p>Full scan spectra from LC-MS profiles of in-gel trypsin digests of α1(I) from human, chicken and xenopus tendon with 6% SDS-PAGE lanes at left (A). MS/MS fragmentation spectrum of the parent ion (1265.3³⁺) from human tendon (B). The sequence is shown with b and y ion breakages. P*, 4Hyp; P#, 3Hyp; K*, Hyl. The cross-linking telopeptide Lys of the human peptide was fully hydroxylated in all posttranslational variants.</p

Mass spectra showing prolyl 3-hydroxylation distributed throughout the whole fibril.

<p>Collagen was solubilized from adult human tendon using SDS extraction (A) and CNBr digestion (B). Lanes of 6% (A) and 12% (B) SDS-PAGE gels are shown to the left. Similar levels of 3Hyp (∼two 3Hyp per α2(I) chain) were observed using each approach. The 1210²⁺ and 1218²⁺ ions in both ion ladders represent unrelated peptides with a 2+ charge (these ions are indicated with φ).</p

Developmental control of prolyl 3-hydroxylation in tendon.

<p>Reduced levels of 3Hyp were observed in fetal tendon relative to adult tissue. MS scan of fetal human Achilles tendon α1(I) with 6% SDS-PAGE lane at left (A). The 1248.4³⁺ ion contains a mix of two peptide posttranslational variants (one 3Hyp and four 4Hyp; and no 3Hyp and five 4Hyp). MS scan of fetal human Achilles tendon α2(I) (B).</p

Summary of 3Hyp occupancy in the (GPP)_n of type I and II collagen α-chains.

<p>The table shows the average number of 3Hyp residues per (GPP)_n motif with the percentage of α-chains containing at least one 3Hyp residue per (GPP)_n given in parentheses. The percentage of each posttranslational variant was determined based on the ratio of the heights of the m/z peaks. For example, the human tendon α1(I) (GPP)_n tryptic peptide, TGDAGPV<b>GPPGPPGPPGPPGPP</b>SAGFDFSFLPQPPQE<b>K</b>, was found to be a mix of eight distinct molecular species giving a hydroxylation (±16 Da) ladder, each representing a posttranslational variant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093467#pone-0093467-g002" target="_blank">Figure 2</a>). The molecular location of the each hydroxylated residue (3Hyp, 4Hyp and Hyl) was determined using MS/MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093467#pone.0093467.s001" target="_blank">Figure S1</a>). The C-terminal lysine was predominantly hydroxylated in all Achilles tendons. In this scroll, the 1270.7³⁺ m/z (peptide species containing four 3Hyp residues and five 4Hyp) represents 9% of the total population and the other variations are as follows: 1265.9³⁺ (three 3Hyp residues and five 4Hyp, 10%); 1260.5³⁺ (three 3Hyp residues and four 4Hyp, 13%); 1254.6³⁺ (two 3Hyp residue and four 4Hyp, 19%); 1249.6³⁺ (one 3Hyp residue and four 4Hyp, 16%); 1244.1³⁺ (no 3Hyp residues and four 4Hyp, 18%); 1238.6³⁺ (no 3Hyp residues and three 4Hyp residue, 10%); 1233.1³⁺ (no 3Hyp residues and two 4Hyp residue, 5%). From these percentages, the average number of 3Hyp residues was estimated per α-chain. In this example the calculation is (4×9%)+(3×10%)+(3×13%)+(2×19%)+(1×16%)  =  mean content of 1.6 3Hyp per α1(I) from human tendon. The 3Hyp content in mouse tendon type I collagen was observed to vary markedly with animal age, in the range between one and two 3Hyp residues per (GPP)_n as indicated in the table.</p