We show that while the structure of the monomer is similar to that of the pentameric core, its Cμ4 domains undergo dynamic separation, which may be relevant to IgM function as the membrane-bound B cell receptor.Ī Sharpened cryo-EM map of FL-IgM showing the high-resolution core structure (threshold 0.6). In comparison, we study the structure of the IgM Fc monomer by both cryo-EM and time-resolved single-molecule fluorescence resonance energy transfer (smFRET). We resolve an F(ab’) 2 at one position of the pentamer, which is stabilised with respect to the core, and interpret disordered density distributions at other Fab positions in terms of a hinge-equivalent motion of IgM. The pentameric core serves as a high molecular weight scaffold to study the structure of the flexibly-attached Fab regions. In this study, we have addressed these questions by analysing the 3D structure of human FL-IgM using a single particle cryo-EM approach, placing the emphasis on understanding the hinge structure and flexibility of IgM. However, where exactly the flexibility comes from, whether at the N- or C-terminus of the Cμ2 domain or both is still ambiguous. Evidence of flexibility between the Fc and Fab fragments of IgM have been shown in several studies, where projections of IgM alone or ligand-bound forms were captured in negative stain EM images 7, 26, 27. This does not necessarily imply that IgM lacks flexibility at this region, and the two potential origins of flexibility are the interdomain regions between Cμ1 and Cμ2, and between Cμ2 and Cμ3. However, little sequence similarity has been found at the hinge-equivalent region of IgM owing to the substitution of an additional Fc domain Cμ2, which has led to the conclusion that IgM lacks a hinge region 24, 25. Traditionally, structures of the hinge regions of immunoglobulin molecules have been identified by enzymatic fragmentations and sequence homology, according to which IgG and IgA are classified into several subclasses 21, 22, 23.

Nevertheless, important questions such as how the peripheral domains are positioned and move with respect to the Fcμ core remain to be answered, which can only be achieved by imaging the structure of FL-IgM as a whole. Individual Cμ4, Cμ3, Cμ2 and Fab domains have been resolved by X-ray crystallography and NMR 11, 12, 13, 14, 15, 16, 17, complexes of IgM with complement components C1 and C4b have been studied at low resolution by electron cryotomography 18, and two recent publications revealed the atomic structure of the asymmetric pentameric Cμ4-Cμ3 core by single-particle cryo-EM 19, 20. As the very first antibody produced after pathogen invasion, the multivalent nature of polymeric IgM molecules gives them remarkable ability to agglutinate particulate antigens in serum 8, 9, 10.Įfforts have been made for decades to study the structure of IgM but have been hindered by its size and flexibility. An IgM pentamer is over 900 kDa, consisting of ten heavy (μ) chains, each of which has a C-terminal tailpiece which holds them together, ten light (λ or κ) chains and a joining (J) chain, a 15 kDa polypeptide with no homology to other proteins, which sterically interrupts the addition of a sixth heavy crystal fragment (Fcμ) unit 5, 6, 7. IgM hexamers have also been found in the serum, but account for less than 5% of total IgM 4. Pentameric IgM is secreted into serum, or transported across epithelial cells by the polymeric immunoglobulin receptor (pIgR) 2, 3. Monomeric IgM serves as a membrane-associated antigen-recognition receptor on the surface of B lymphocytes 1. Natural immunoglobulin M (IgM) assembles into monomers (composed of two heavy and two light chains), pentamers and hexamers. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cμ4 domain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cμ3) at the IgM subunit interacting most extensively with the J-chain. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently.

A hinge is located at the Cμ3/Cμ2 domain interface, allowing Fabs and Cμ2 to pivot as a unit both in-plane and out-of-plane. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cμ4 and Cμ3 constant regions and the J-chain. Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens.

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