Docking Server for the Identification of Heparin Binding Sites on Proteins

Many proteins of widely differing functionality and structure are capable of binding heparin and heparan sulfate. Since crystallizing protein–heparin complexes for structure determination is generally difficult, computational docking can be a useful approach for understanding specific interactions. Previous studies used programs originally developed for docking small molecules to well-defined pockets, rather than for docking polysaccharides to highly charged shallow crevices that usually bind heparin. We have extended the program PIPER and the automated protein–protein docking server ClusPro to heparin docking. Using a molecular mechanics energy function for scoring and the fast Fourier transform correlation approach, the method generates and evaluates close to a billion poses of a heparin tetrasaccharide probe. The docked structures are clustered using pairwise root-mean-square deviations as the distance measure. It was shown that clustering of heparin molecules close to each other but having different orientations and selecting the clusters with the highest protein–ligand contacts reliably predicts the heparin binding site. In addition, the centers of the five most populated clusters include structures close to the native orientation of the heparin. These structures can provide starting points for further refinement by methods that account for flexibility such as molecular dynamics. The heparin docking method is available as an advanced option of the ClusPro server at http://cluspro.bu.edu/

A Novel Dimeric Inhibitor Targeting Beta2GPI in Beta2GPI/Antibody Complexes Implicated in Antiphospholipid Syndrome

Background: b2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of b2GPI generated by anti-b2GPI antibodies is pathologically important, in contrast to monomeric b2GPI which is abundant in plasma. Principal Findings: We created a dimeric inhibitor, A1-A1, to selectively target b2GPI in b2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of b2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of b2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of b2GPI present in human serum, b2GPI purified from human plasma and the individual domain V of b2GPI. We demonstrated that when b2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of b2GPI to cardiolipin, regardless of the source of b2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of b2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-b2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric b2GPI to cardiolipin. Conclusions: Our results suggest that the approach of using a dimeric inhibitor to block b2GPI in the pathologica

Solution structure and dynamics of bioactive peptides mimicking NGF

Nerve growth factor (NGF) belongs to the neurotrophin family of growth factors. NGF can bind to two transmembrane receptors, TrkA and p75. TrkA is the NGF specific tyrosine kinase receptor; p75 is a receptor which is common to all neurotrophins. NGF is required for the development, maintenance and repair of the nervous system. NGF dependent neuronal populations are affected in many diseases and the use of NGF was proposed. Difficulties associated with therapeutic application of proteins and polypeptides could be overcome by the use of small peptidomimetics. Small molecule that can either mimic or inhibit NGF-TrkA signaling could be useful in therapy of a wide range of pathologies from neurodegeneration to cancer.Peptide mimetics represent an intermediate step towards the development of potent drugs. Small NGF mimetics could be found either in random peptide libraries or by rational drug design. Rational design is based on structure-function knowledge about the binding interface. We approach the problem by designing a series of cyclic peptides from the C-D loop of NGF that is known to interact with TrkA receptor. Nuclear Magnetic Resonace (NMR) was used to determine the structure and dynamics of the peptides in solution. The structural information was combined with bioactivity tests. Comparison of the structures revealed close similarity between the peptides and the conformation of the C-D loop in X-ray crystal structures of NGF. Structural similarity ensures an appropriate backbone conformation and orientation of the sidechains involved in interactions with receptor. This work contributes to our understanding of the structure-function relationships in NGF-TrkA binding. The designed bioactive peptides could serve as a template for further design of smaller peptidomimetics of NGF with improved potency and stability. A similar approach could be used to design functional mimetics of the other neurotrophins

Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops

Upregulation of the procoagulant activity of monocytes by antibodies to beta2-glycoprotein I (β2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by β2GPI/anti-β2GPI complexes and their binding sites on β2GPI is far from complete. We quantified the procoagulant activity expressed by phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells by measuring clotting kinetics in human plasma exposed to stimulated cells. Cells stimulated with anti-β2GPI were compared to cells treated with dimerized domain V of β2GPI (β2GPI-DV) or point mutants of β2GPI-DV. We demonstrated that dimerized β2GPI-DV is sufficient to induce procoagulant activity in monocytes. Using site-directed mutagenesis, we determined that the phospholipid-binding interface on β2GPI is larger than previously thought and includes Lys308 in β2GPI-DV. Intact residues in two phospholipid-binding loops of β2GPI-DV were important for the potentiation of procoagulant activity. We did not detect a correlation between the ability of β2GPI-DV variants to bind ApoER2 and potentiation of the procoagulant activity of cells. The region on β2GPI inducing procoagulant activity in monocytes can now be narrowed down to β2GPI-DV. The ability of β2GPI-DV dimers to come close to cell membrane and attach to it is important for the stimulation of procoagulant activity

Solution structure and internal motion of a bioactive peptide derived from nerve growth factor

NRC publication: Ye

Design and solution structure of functional peptide mimetics of nerve growth factor

NRC publication: Ye

Comparison of the inhibition of β2GPI in serum with the inhibition of the isolated domain V by monomeric A1 in the absence of dimerization antibodies.

<p>Inhibition of the binding of β2GPI-DV by A1 (circles); inhibition of the binding of β2GPI in serum by A1 (triangles).</p

Inhibition of the binding of β2GPI in human serum to cardiolipin in the absence of anti-β2GPI antibodies.

<p>Inhibition curves measured for the dimeric inhibitor A1-A1 (circles) and monomeric inhibitor A1 (triangles). On the plot, the data points at 50 µM and 100 µM of A1-A1 partially overlap with the corresponding data points of A1. To facilitate comparison, the measured OD₄₀₅ values and the binding curves were normalized to the maximum binding obtained from the fit of the raw data to a one-site inhibition model.</p

Binding and inhibition of the binding of β2GPI in normal human serum to cardiolipin.

<p>A) The binding of β2GPI in serum to cardiolipin-coated surface in the absence (triangles) and in the presence (circles) of anti-β2GPI antibodies. B) Inhibition of the binding of β2GPI in human serum to cardiolipin in the presence of anti-β2GPI antibodies by the dimeric inhibitor A1-A1 (circles) and monomeric inhibitor A1 (triangles). Fit to one-site binding and inhibition models was performed on the raw data. To facilitate comparison, the measured OD₄₀₅ values and the binding curves were normalized to the maximum binding obtained from the fit.</p