Reha Celikel

von Willebrand factor conformation and adhesive function is modulated by an internalized water molecule.

Platelet participation in hemostasis and arterial thrombosis requires the binding of glycoprotein (GP) Ibα to von Willebrand factor (vWF). Hemodynamic forces enhance this interaction, an effect mimicked by the substitution I546V in the vWF A1 domain. A water molecule becomes internalized near the deleted Ile methyl group. The change in hydrophobicity of the local environment causes positional changes propagated over a distance of 27 Å. As a consequence, a major reorientation of a peptide plane occurs in a surface loop involved in GP Ibα binding. This distinct vWF conformation shows increased platelet adhesion and provides a structural model for the initial regulation of thrombus formation.

Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIbα-N. Mutating Tyr276, which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr278 or Tyr279, which mostly contact exosite I residues, reduced FIIa complexing in solution by 0–20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands—aptamer HD1, hirugen, and lepirudin—did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp148 orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys279. These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti‐(+)‐METH therapeutic single chain antibody fragment (scFv6H4, KD= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4‐methylenedioxymethamphetamine (MDMA), known by the street name “ecstasy.” The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a CH ⃛O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 Å, and 2.4 Å, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering

Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP.

Platinum-induced space-group transformation in crystals of the platelet glycoprotein Ibα N-terminal domain

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for thrombus formation, leading to the arrest of bleeding. The N-terminal domain of GP Ib alpha, which contains the binding sites for VWF and alpha-thrombin, crystallized in the tetragonal space group P4(3) with one molecule in the asymmetric unit. When the crystals were treated with platinum, the crystals changed their symmetry from tetragonal to monoclinic P2(1) with two molecules in the asymmetric unit. The structure of the monoclinic form was solved using two-wavelength platinum anomalous dispersion data. The tetragonal crystal structure was subsequently solved using molecular-replacement techniques using the monoclinic structure as the search model and was refined with 1.7 A resolution data.

Structure and Function of the Von Willebrand Factor A1 Domain

The role of von Willebrand factor (VWF) in blocking hemorrhage is centered on its ability to act as a bridging adhesive molecule between platelets and components of the extracellular matrix or other platelets. In the course of chronic vascular diseases, moreover, the same properties of VWF may become the cause of pathological thrombus formation leading to arterial occlusion. There is convincing evidence that VWF functions involving interactions with platelets ultimately depend on binding to the membrane glycoprotein (GP) Ibalpha receptor mediated by the A1 domain. In this review, we present the current knowledge on the structural features of the VWF A1 domain that support its functions.

Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites.

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, KD = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.

175 Structure-based engineering to generate high-affinity immunotherapy for the drug of abuse

Methamphetamine (METH) abuse is a major threat in the USA and worldwide without any FDA approved medications. Anti-METH antibody antagonists block or slow the rate of METH entry into the brain and have shown efficacy in preclinical studies (Peterson, Laurenzana, Atchley, Hendrickson, & Owens, 2008). A key determinant of the antibody’s efficacy is its affinity for METH and we attempted to enhance the efficacy by designing mutations to alter the shape or the electrostatic character of the binding pocket. Towards this goal, we developed a single chain anti-METH antibody fragment (scFv6H4) from a parent IgG (1). The crystal structure of scFv-6H4 in complex with METH was determined (Celikel, Peterson, Owens, & Varughese, 2009). Based on its elucidated binding interactions, we designed point mutations in the binding pocket to improve its affinity for METH and amphetamine (AMP), the active metabolite of METH. The mutants, scFv-S93T,-I37 M and -Y34 M were cloned, expressed in yeast and tested for affinity against METH and AMP. Two mutants showed enhanced binding affinity for METH: scFv-I37 M by 1.3-fold and scFv-S93T by 2.6-fold. Additionally, all the mutants showed increase in affinity for AMP: scFv-I37 M by 56-fold, scFv-S93T by 17-fold and scFvY34 M by 5-fold. Crystal structure for one of the high-affinity mutant, scFv-S93T, in complex with METH was determined (Figure 1). Binding pocket of the mutant is more hydrophobic in comparison with the wild type. ScFv-6H4 binds METH in a deep pocket containing two water molecules. The substitution of a serine residue by a threonine leads to the expulsion of a water molecule (Figure 2), relieving some unfavorable contacts between the hydrocarbon atoms of METH and the water molecule and increasing the affinity to sub-nanomolar range. Therefore, the present study shows that efficacy could be enhanced by altering the hydrophobicity or the shape of the binding pocket.