performed HDX-MS experiments and analyses; H.W. Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcRIIIa, in addition to GPR120 modulator 1 the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering. Subject terms: Molecular biophysics, Atomic force microscopy Introduction The combination of antigen recognition and expression of effector functions typified by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity is a major function of the antibody1C4. To exert this hub function, the antibody structure is divided into Fab arms and Fc stem. The Fab portions exhibit sequence variability in the N-terminal domains VH and VL, which recognize various antigens, followed by constant domains CL and CH1. By contrast, the Fc region has a two-fold-symmetric homodimeric structure comprising two CH2 and two CH3 domains. The relative orientation of the Fab arms with respect to the Fc stem varies because of their connection through a flexible linker called a hinge1,5. The Fc portion of immunoglobulin G (IgG) provides interaction sites for effector molecules such as complement1C4. Most of the cells working in the immune system express receptors for IgG, possessing extracellular regions comprising Ig-fold domains and interacting with the Fc portion of IgG6C11. Therefore, they are collectively termed Fc receptors (FcRs). FcRs are classified into three major isoforms: FcRI, FcRII, and FcRIII, each exhibiting different binding affinities to the IgG isotypes, and distinct expression profiles on immune cells12C14. Human FcRIII is further divided into two isoformstransmembrane FcRIIIa and glycosylphosphatidylinositol-linked FcRIIIbthat share 96% amino acid sequence identity in their extracellular regions. FcRIIIa, expressed primarily on natural killer cells, promotes ADCC by interacting with the IgG in complex with antigen, whereas FcRIIIb, expressed exclusively on neutrophils, mediates the degranulation and phagocytosis of the IgG-labeled target cells15C19. The interaction modes of human IgG1 and FcRIII molecules have been structurally characterized by X-ray crystallography using the Fc fragments and the soluble forms of FcRIII (sFcRIII) molecules, comprising the D1 and D2 domains20C25. These studies have identified their primary interaction sitesnamely, the hinge-proximal segments in the CH2 domains of Fc and the loops in the membrane-proximal D2 domain in FcRsand also revealed the domain rearrangements in both proteins. Furthermore, the practical significance of intermolecular carbohydrate-carbohydrate relationships has been underscored in the GPR120 modulator 1 connection between human being IgG1-Fc and the extracellular region of FcRIIIa, considered to be probably one of the most crucial factors for medical applications of human being IgG1-based restorative antibodies that target cancers22,23,26C28. However, a large gulf exists between the structural views therefore obtained and the practical and restorative insights gained from observations under physiologically practical conditions where IgG1 interacts with FcRIIIa anchored within the cell surfaces through a C-terminal transmembrane section. Here, using high-speed atomic pressure microscopy (HS-AFM) and human being, humanized, and mouse/human-chimeric IgG1 antibodies (Supplementary Fig.?1) and their Fc fragments, along Goat polyclonal to IgG (H+L)(Biotin) with human being sFcRIIIa immobilized through its C-terminal section within the scanning surface, we perform real-time observation of IgG-FcR GPR120 modulator 1 relationships. The connection modes of these IgGs with sFcRIIIa have also been characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) in answer. Results HS-AFM observations of IgG1-FcRIIIa We prepared a recombinant sFcRIIIa glycoprotein having a C-terminal hexahistidine moiety for immobilization onto a Ni2+-coated mica surface. When the IgG answer was added to the observation buffer in the sample chamber of HS-AFM, IgG molecules went to the extracellular domains of FcRIIIa, transiently forming a 1:1 complex (Supplementary Video clips?1C3). We compared interactions of a panel of IgGs by HS-AFM imaging (Fig.?1a) by quantifying dwell occasions of the IgG molecules and summarize them in Fig.?2. The human being, humanized, and mouse/chimeric IgG1 antibodiesPMF37, trastuzumab, and rituximab, respectivelyexhibited similar dwell occasions of around GPR120 modulator 1 1?s. Open in a separate window Number 1 HS-AFM observations of relationships between IgG1 antibodies and their Fc fragments with sFcRIIIa. (a) We indicate full-length rituximab by a reddish arrow (Supplementary Video clips?1C3). (b) We indicate rituximab Fc by a magenta arrow (Supplementary Video clips?4C6). In (a,b), we display an immobilized sFcRIIIa molecule having two extracellular domains by a white arrow. Open in a separate window GPR120 modulator 1 Number 2 Dwell occasions of three kinds of IgG1 and their Fc fragments on sFcRIIIa. We subjected the Fc fragments derived from these IgGs to HS-AFM observation (Fig.?1b, Supplementary Video clips?4C6). Unexpectedly, dwell occasions of the Fc fragments were remarkably decreased (0.31 sC0.35?s) in comparison with those of the intact IgGs (Fig.?2). We confirmed that mock-treated control (PMF37 incubated in the absence of papain) managed the longer dwell time (Supplementary Fig.?2). These data show the Fab portions of IgG contribute positively to its connection with FcRIIIa. HDX-MS characterization of IgG1-FcRIIIa connection The HS-AFM observation raised the possibility of direct involvement of the Fab portion of IgG1 in the connection with sFcRIIIa..