Mark Agostino

Challenges and advances in computational docking: 2009 in review

Docking is a computational technique that places a small molecule (ligand) in the binding site of its macromolecular target (receptor) and estimates its binding affinity. This review addresses methodological developments that have occurred in the docking field in 2009, with a particular focus on the more difficult, and sometimes controversial, aspects of this promising computational discipline. These developments aim to address the main challenges of docking: receptor representation (such aspects as structural waters, side chain protonation, and, most of all, flexibility (from side chain rotation to domain movement)), ligand representation (protonation, tautomerism and stereoisomerism, and the effect of input conformation), as well as accounting for solvation and entropy of binding. This review is strongly focused on docking advances in the context of drug design, specifically in virtual screening and fragment‐based drug design. Copyright

Molecular Docking of Carbohydrate Ligands to Antibodies: Structural Validation against Crystal Structures

Cell surface glycoproteins play vital roles in cellular homeostasis and disease. Antibody recognition of glycosylation on different cells and pathogens is critically important for immune surveillance. Conversely, adverse immune reactions resulting from antibody-carbohydrate interactions have been implicated in the development of autoimmune diseases and impact areas such as xenotransplantation and cancer treatment. Understanding the nature of antibody-carbohydrate interactions and the method by which saccharides fit into antibody binding sites is important in understanding the recognition process. In silico techniques offer attractive alternatives to experimental methods (X-ray crystallography and NMR) for the study of antibody-carbohydrate complexes. In particular, molecular docking provides information about protein-ligand interactions in systems that are difficult to study with experimental techniques. Before molecular docking can be used to investigate antibody-carbohydrate complexes, validation of an appropriate docking method is required. In this study, four popular docking programs, Glide, AutoDock, GOLD, and FlexX, were assessed for their ability to accurately dock carbohydrates to antibodies. Comparison of top ranking poses with crystal structures highlighted the strengths and weaknesses of these programs. Rigid docking, in which the protein conformation remains static, and flexible docking, where both the protein and ligand are treated as flexible, were compared. This study has revealed that generally molecular docking of carbohydrates to antibodies has been performed best by Glide.

In silico analysis of antibody–carbohydrate interactions and its application to xenoreactive antibodies

Antibody-carbohydrate interactions play central roles in stimulating adverse immune reactions. The most familiar example of such a process is the reaction observed in ABO-incompatible blood transfusion and organ transplantation. The ABO blood groups are defined by the presence of specific carbohydrates expressed on the surface of red blood cells. Preformed antibodies in the incompatible recipient (i.e., different blood groups) recognize cells exhibiting host-incompatible ABO system antigens and proceed to initiate lysis of the incompatible cells. Pig-to-human xenotransplantation presents a similar immunological barrier. Antibodies present in humans recognize carbohydrate antigens on the surface of pig organs as foreign and proceed to initiate hyperacute xenograft rejection. The major carbohydrate xenoantigens all bear terminal Gal alpha(1,3)Gal epitopes (or alphaGal). In this study, we have developed and validated a site mapping technique to investigate protein-ligand recognition and applied it to antibody-carbohydrate systems. This site mapping technique involves the use of molecular docking to generate a series of antibody-carbohydrate complexes, followed by analysis of the hydrogen bonding and van der Waals interactions occurring in each complex. The technique was validated by application to a series of antibody-carbohydrate crystal structures. In each case, the majority of interactions made in the crystal structure complex were able to be reproduced. The technique was then applied to investigate xenoantigen recognition by a panel of monoclonal anti-alphaGal antibodies. The results indicate that there is a significant overlap of the antibody regions engaging the xenoantigens across the panel. Likewise, similar regions of the xenoantigens interact with the antibodies.

Identification of preferred carbohydrate binding modes in xenoreactive antibodies by combining conformational filters and binding site maps

Carbohydrates are notoriously flexible molecules. However, they have an important role in many biochemical processes as specific ligands. Understanding how carbohydrates are recognized by other biological macromolecules (usually proteins) is therefore of considerable scientific value. Interfering with carbohydrate-protein interactions is a potentially useful strategy in combating a range of disease states, as well as being of critical importance in facilitating allo- and xenotransplantation. We have devised an in silico protocol for analyzing carbohydrate-protein interactions. In this study, we have applied the protocol to determine the structures of alphaGal-terminating carbohydrate antigens in complex with a panel of xenoreactive antibodies. The most important feature of the binding modes is the fixed conformation of the Galbeta(1,4)Glc/GlcNAc linkage across all of the binding modes. The preferred conformation of the terminal Galalpha(1,3)Gal linkage varies depending on the antibody binding site topography, although it is possible that some of the antibodies studied recognize more than one Galalpha(1,3)Gal conformation. The binding modes obtained indicate that each antibody uses distinct mechanisms in recognizing the target antigens.

Antibody Recognition of Cancer-Related Gangliosides and Their Mimics Investigated Using in silico Site Mapping

Modified gangliosides may be overexpressed in certain types of cancer, thus, they are considered a valuable target in cancer immunotherapy. Structural knowledge of their interaction with antibodies is currently limited, due to the large size and high flexibility of these ligands. In this study, we apply our previously developed site mapping technique to investigate the recognition of cancer-related gangliosides by anti-ganglioside antibodies. The results reveal a potential ganglioside-binding motif in the four antibodies studied, suggesting the possibility of structural convergence in the anti-ganglioside immune response. The structural basis of the recognition of ganglioside-mimetic peptides is also investigated using site mapping and compared to ganglioside recognition. The peptides are shown to act as structural mimics of gangliosides by interacting with many of the same binding site residues as the cognate carbohydrate epitopes. These studies provide important clues as to the structural basis of immunological mimicry of carbohydrates.

Free Ig Light Chains Interact with Sphingomyelin and Are Found on the Surface of Myeloma Plasma Cells in an Aggregated Form

Free κ L chains (FκLCs) are expressed on the surface of myeloma cells and are being assessed as a therapeutic target for the treatment of multiple myeloma. Despite its clinical potential, the mechanism by which FκLCs interact with membranes remains unresolved. In this study, we show that FκLCs associate with sphingomyelin on the plasma membrane of myeloma cells. Moreover, membrane-bound FκLCs are aggregated, suggesting that aggregation is required for intercalation with membranes. Finally, we propose a model where the binding of FκLCs with sphingomyelin on secretory vesicle membranes is stabilized by self-aggregation, with aggregated FκLCs exposed on the plasma membrane after exocytosis. Although it is well known that protein aggregates bind membranes, this is only the second example of an aggregate being found on the surface of cells that also secrete the protein in its native form. We postulate that many other aggregation-prone proteins may associate with cell membranes by similar mechanisms.

Carbohydrate-mimetic peptides: structural aspects of mimicry and therapeutic implications

Introduction: The existence of specific carbohydrates on the surface of a wide range of cells provides the opportunity for the development of highly targeted therapeutic agents. The potential applications of such agents are diverse, and include vaccines against pathogenic microorganisms, cancer and HIV, and anti-rejection agents for organ transplantation. However, the use of carbohydrates as either therapeutic agents or immunogens is frequently problematic, as they are often rapidly metabolized and poorly immunogenic. Therefore, the search for carbohydrate-mimetic agents is of considerable therapeutic value, for the potential of such agents to both interfere with carbohydrate–protein interactions and to generate carbohydrate-specific immune responses. Areas covered: The review discusses recent examples of carbohydrate-mimetic peptides with regard to the structural and functional aspects of mimicry and the implications of peptide mimicry for application in therapeutics. The reader will gain knowledge of the various mechanisms of peptide carbohydrate mimicry, and the potential importance of these mechanisms in targeted therapeutic design. Expert opinion: Peptide carbohydrate mimicry is manifested by distinct mechanisms, any one of which may be relevant to specific protein targets. As structural information becomes available for a wider variety of systems, the questions about mimicry will be more effectively addressed.

Structural biology of carbohydrate xenoantigens

Transplantation of organs across species (xenotransplantation) is being considered to overcome the shortage of human donor organs. However, unmodified pig organs undergo an antibody-mediated hyperacute rejection that is brought about by the presence of natural antibodies to Galα(1,3)Gal, which is the major carbohydrate xenoantigen. Genetic modification of pig organs to remove most of the Galα(1,3)Gal epitopes has been achieved, but the human immune system may still recognize residual lipid-linked Galα(1,3)Gal carbohydrates, new (cryptic) carbohydrates or additional non-Galα(1,3)Gal carbohydrate xenoantigens. The structural basis for lectin and antibody recognition of Galα(1,3)Gal carbohydrates is starting to be understood and is discussed in this review. Antibody binding to Galα(1,3)Gal carbohydrates is predicted to primarily involve end-on insertion of the terminal αGal residue, but it is possible that groove-type binding can occur, as for some lectins. It is likely that similar antibody and lectin recognition will occur with other non-Galα(1,3)Gal xenoantigens, which potentially represent new barriers for pig-to-human xenotransplantation.

A computational approach for exploring carbohydrate recognition by lectins in innate immunity

Recognition of pathogen-associated carbohydrates by a broad range of carbohydrate-binding proteins is central to both adaptive and innate immunity. A large functionally diverse group of mammalian carbohydrate-binding proteins are lectins, which often display calcium-dependent carbohydrate interactions mediated by one or more carbohydrate recognition domains. We report here the application of molecular docking and site mapping to study carbohydrate recognition by several lectins involved in innate immunity or in modulating adaptive immune responses. It was found that molecular docking programs can identify the correct carbohydrate-binding mode, but often have difficulty in ranking it as the best pose. This is largely attributed to the broad and shallow nature of lectin binding sites, and the high flexibility of carbohydrates. Site mapping is very effective at identifying lectin residues involved in carbohydrate recognition, especially with cases that were found to be particularly difficult to characterize via molecular docking. This study highlights the need for alternative strategies to examine carbohydrate–lectin interactions, and specifically demonstrates the potential for mapping methods to extract additional and relevant information from the ensembles of binding poses generated by molecular docking.

Development and application of site mapping methods for the design of glycosaminoglycans.

Glycosaminoglycans (GAGs) are complex polysaccharides involved in a wide range of biological signaling events, as well as being important as biological structural materials. Despite the ubiquity and importance of GAG-protein interactions in biological systems and potentially as therapeutic targets, detailed structures of such interactions are sparse in availability. Computational methods can provide detailed structural knowledge of these interactions; however, they should be evaluated against suitable test systems prior to their widespread use. In this study, we have investigated the application of automated molecular docking and interaction mapping techniques to characterizing GAG-protein interactions. A series of high-resolution X-ray crystal structures of GAGs in complex with proteins was used to evaluate the approaches. Accurately scoring the pose fitting best with the crystal structure was a challenge for all docking programs evaluated. The site mapping technique offered excellent prediction of the key residues involved in ligand recognition, comparable to the best pose and improved over the top-ranked pose. A design protocol incorporating site- and ligand-based mapping techniques was developed and applied to identify GAGs capable of binding to acidic fibroblast growth factor (aFGF). The protocol was able to identify ligands known to bind to aFGF and accurately able to predict the binding modes of those ligands when using a known ligand-binding conformation of the protein. This study demonstrates the value of mapping-based techniques in identifying specific GAG epitopes recognized by proteins and for GAG-based drug design.