The human IgM pentamer is a mushroom-shaped molecule with a flexural bias

Abstract

The textbook planar model of pentameric IgM, a potent activator of complement C1q, is based upon the crystallographic structure of IgG. Although widely accepted, key predictions of this model have not yet been directly confirmed, which is particularly important since IgG lacks a major Ig fold domain in its Fc region that is present in IgM. Here, we construct a homology-based structural model of the IgM pentamer using the recently obtained crystallographic structure of IgE Fc, which has this additional Ig domain, under the constraint that all of the cysteine residues known to form disulfide bridges both within each monomer and between monomers are bonded together. In contrast to the planar model, this model predicts a non-planar, mushroom-shaped complex, with the central portion formed by the C-terminal domains protruding out of the plane formed by the Fab domains. This unexpected conformation of IgM is, however, directly confirmed by cryo-atomic force microscopy of individual human IgM molecules. Further analysis of this model with free energy calculations of out-of-plane Fab domain rotations reveals a pronounced asymmetry favoring flexions toward the central protrusion. This bias, together with polyvalent attachment to cell surface antigen, would ensure that the IgM pentamer is oriented on the cell membrane with its C1q binding sites fully exposed to the solution, and thus provides a mechanistic explanation for the first steps of C1q activation by IgM.AFM ͉ homology modeling ͉ immunoglobulin ͉ single molecule P entameric IgM is an important component of the first line of defense against foreign pathogens (1, 2) and possibly modified self-components (3), and is increasingly being developed in the diagnosis and therapy of malignancies (4-7). It is also implicated in the damage to organs and tissues following ischemia/reperfusion (8) and in multiple autoimmune diseases (9-11). Its best understood mechanism of action in the immune response is as the initiating component in the classical complement pathway mediated by C1q (12). In this, the binding of IgM to cell surface antigen enables C1q to bind to IgM, which thereby activates C1q for interactions with downstream components. The expected structural changes consequent to antigen-binding and associated with C1q activation, and how these might be modified in disease-conditions, have spurred a longstanding interest in the structure of this large multicomponent molecule (13-17).Like other antibodies, IgM monomers consist of two light and two heavy chains. However, whereas the heavy chains of most antibodies (such as IgG) contain three constant Ig domains, the heavy chains of IgM have a fourth one, as do the heavy chains in IgE. These extra (C 2) domains are located in place of the proline-rich hinge region that is responsible for the rotational flexibility of the antigen-binding Fab domains (relative to the Fc domain).Five IgM monomers complex with an additional small polypeptide (the J chain) to form the predominant oligomeric species in the human plasma (3). Obtaini...

High-resolution structures of the IgM Fc domains reveal principles of its hexamer formation

“…4C). The dimensions of this structure are in good agreement with previous estimations based on cryo-atomic force microscopy (AFM) data (13). In addition to the nonplanar shape, also the size of the central circular region, composed of the Cμ3 and Cμ4 domains, with a diameter of ∼180 Å, fits well to the core region dimension of 190 ± 20 Å seen in the cryo-AFM experiments.…”

“…A detailed mutagenesis study showed that the cysteine at position 575 is essential for efficient assembly, whereas C337 and C414 are not needed for polymer formation (24). The latest IgM model is based on the IgE structure (13).…”

“…NMR data were acquired at 25°C on a Bruker Avance III 600 MHz spectrometer with a 300-μM uniformly 15 N, 13 C-labeled Cμ3C414S sample except for the stereospecific assignment of valine and leucine side chains, which was based on a 1 H-13 C heteronuclear multiple quantum correlation acquired on a 10% 13 C-labeled sample as previously described (37,38). Details are provided in SI Materials and Methods.…”

“…Fluorescence data (12) and the subsequently determined crystal structure (11) of IgE Fc revealed that the IgE Fc is sharply bent. Interestingly, the latest IgM model suggests a similar Fc region with the Cμ4 domains protruding out of the plane defined by Cμ2, Cμ3, and the Fab domains (13).…”

Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair

“…It is well established that polyvalency, the presentation of multiple bioactive binding sites, significantly enhances binding strength for biological interactions, a phenomenon known as avidity (38). This phenomenon has a natural basis, vital to the function of binding proteins such as IgM with 10 binding sites (39) or the biological adhesion of many viruses (40), and even the extracellular matrix is polyvalent (41). Polyvalency is used frequently in the design of synthetic bioactive molecules to enhance their binding strength and has been used in the creation of bioactive peptides, organic molecules, carbohydrates, nucleotides, antibiotics, and phage mimics, among others (42)(43)(44)(45)(46).…”