Antibody modeling using the Prediction of ImmunoGlobulin Structure (PIGS) web server

Marcatili, Paolo; Olimpieri, Pier Paolo; Chailyan, Anna; Tramontano, Anna

doi:10.1038/nprot.2014.189

Cited by 56 publications

(64 citation statements)

References 80 publications

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“…HADDOCK docking was performed using the lowest energy structure from the BAX NMR structural ensemble from the PDB (PDB code 1F16). The 3G11 structural model was generated using prediction of immunoglobulin structure software on the basis of structural homology modeling of known Fab structures of the same family that have different amino acid compositions of CDR regions (32). Calculations were performed with ambiguous interaction restraints derived from the hydrogen-deuterium exchange mass spectrometry data and residues of the CDR regions of 3G11.…”

Section: Methodsmentioning

confidence: 99%

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

Uchime

Dai

Biris

et al. 2016

Journal of Biological Chemistry

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The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices ␣1/␣6 and the ␣1-␣2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation.Apoptosis plays a critical role in maintaining normal tissue homoeostasis in multicellular organisms, and its deregulation results in an imbalance of homeostasis contributing to several diseases (1, 2) The BCL-2 protein family plays a central role in regulating the mitochondrial pathway of apoptosis (3, 4). Mitochondrial outer membrane permeabilization (MOMP) 5 is considered the key event that is regulated by the complex network of protein-protein interactions between pro-apoptotic and anti-apoptotic members of the BCL-2 family. Activation of the pro-apoptotic members Bcl-2-associated X protein (BAX) and/or Bcl2-antagonist/killer 1 (BAK) is required for induction of MOMP, whereas the anti-apoptotic or survival proteins, such as BCL-2, BCL-X L , and MCL-1, inhibit the pro-apoptotic proteins and prevent MOMP. Activation of BAX and BAK or inhibition of anti-apoptotic BCL-2 proteins is regulated by direct interaction with the BH3-only proteins.The activation pathway of BAX represents the gateway to apoptosis, and understanding the function of BAX and its regulation mechanisms is an area of intensive investigation. BAX is found predominantly in the cytosolic compartment in an inactive conformation (5, 6). Upon cellular stress, BAX is triggered and undergoes a series of conformational changes that enable its translocation to the mitochondrial...

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Section: Methodsmentioning

confidence: 99%

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

Uchime

Dai

Biris

et al. 2016

Journal of Biological Chemistry

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“…In addition to RosettaAntibody, there are three publicly accessible, fully automated web servers for antibody structure prediction: Kotai Antibody Builder 46,47 , Prediction of ImmunoGlobulin Structures (PIGS) 48 , and ABodyBuilder 49 . The performance of each method was discussed in the recent Antibody Modeling Assessment (AMA) 6 , except for ABodyBuilder, which was developed and benchmarked on the AMA antibodies ex post facto .…”

Section: Introductionmentioning

confidence: 99%

Modeling and docking of antibody structures with Rosetta

et al. 2017

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We describe Rosetta-based computational protocols for predicting the three-dimensional structure of an antibody from sequence (RosettaAntibody) and then docking the antibody to protein antigens (SnugDock). Antibody modeling leverages canonical loop conformations to graft large segments from experimentally-determined structures as well as (1) energetic calculations to minimize loops, (2) docking methodology to refine the VL–VH relative orientation, and (3) de novo prediction of the elusive complementarity determining region (CDR) H3 loop. To alleviate model uncertainty, antibody–antigen docking resamples CDR loop conformations and can use multiple models to represent an ensemble of conformations for the antibody, the antigen or both. These protocols can be run fully-automated via the ROSIE web server (http://rosie.rosettacommons.org/) or manually on a computer with user control of individual steps. For best results, the protocol requires roughly 1,000 CPU-hours for antibody modeling and 250 CPU-hours for antibody–antigen docking. Tasks can be completed in under a day by using public supercomputers.

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“…By now, a number of in silico tools is available to guide the humanization process 16,17 and to provide automatically generated antibody Fv models. 18 In the following, we focus on an aspect of antibody structure that, in the context of humanization, is often overlooked: the relative orientation of VH and VL domain. A survey of the known repertoire of antibody crystal structures reveals a notable variability in the parameters of VH-VL orientation, [19][20][21] and it seems likely that modulating VH-VL orientation not only is a necessary means to accommodate the diverse antigenic shapes that antibodies are confronted with, but also a mechanism to further diversify the composition (and thus increase the possible number) of antibody paratopes -in addition to the well-known mechanisms of diversification involving variations in length and sequence of the CDRs.…”

Section: Introductionmentioning

confidence: 99%

VH-VL orientation prediction for antibody humanization candidate selection: A case study

Bujotzek¹,

Lipsmeier²,

Harris³

et al. 2015

mAbs

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Antibody humanization describes the procedure of grafting a non-human antibody's complementaritydetermining regions, i.e., the variable loop regions that mediate specific interactions with the antigen, onto a b-sheet framework that is representative of the human variable region germline repertoire, thus reducing the number of potentially antigenic epitopes that might trigger an anti-antibody response. The selection criterion for the so-called acceptor frameworks (one for the heavy and one for the light chain variable region) is traditionally based on sequence similarity. Here, we propose a novel approach that selects acceptor frameworks such that the relative orientation of the 2 variable domains in 3D space, and thereby the geometry of the antigen-binding site, is conserved throughout the process of humanization. The methodology relies on a machine learning-based predictor of antibody variable domain orientation that has recently been shown to improve the quality of antibody homology models. Using data from 3 humanization campaigns, we demonstrate that preselecting humanization variants based on the predicted difference in variable domain orientation with regard to the original antibody leads to subsets of variants with a significant improvement in binding affinity.

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Antibody modeling using the Prediction of ImmunoGlobulin Structure (PIGS) web server

Cited by 56 publications

References 80 publications

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site

Modeling and docking of antibody structures with Rosetta

VH-VL orientation prediction for antibody humanization candidate selection: A case study

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