Figures and data in The structures of secretory and dimeric immunoglobulin A

Figures
Tables
Additional files

7 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement

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SIgA delivery to the mucosa.

(A) Schematic depicting unliganded pIgR binding to dIgA from the lamina propria on the basolateral surface of an epithelial cell followed by transcytosis to the apical membrane and SIgA release into the mucosa. (B) Schematic showing protein components of SIgA, including two IgA monomers, joining chain (JC), and secretory component (SC). The IgA heavy chain is colored white with a black outline and the light chain is colored white with a gray outline. Each chain is made up of immunoglobulin domains, including IgA heavy chain constant (C_H1-3), heavy chain variable (V_H), light chain constant (C_L), and light chain variable (V_L),domains, which are indicated along with antigen-binding fragments (Fabs) and Fc regions.

Figure 1—figure supplement 1

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Sequence identity, similarity, and alignments between mouse and human SIgA components.

(A) Sequence identity and similarity calculated between the indicated mouse and human sequences. (B) Sequences of the mouse SIgA structure components aligned to homologous human sequences. The sequences for individual protein domains are separated by line and labeled; identical residues are highlighted in red. Molecular contacts between JC, SC and each heavy chain observed in the SIgA structure are indicated by squares. Each square denotes an interfacing residue described in the main text and/or within 4 Å of a residue on another chain. Squares are colored according to the key at the bottom of the figure; contacts occurring between the four heavy chains are not indicated.

Figure 2 with 4 supplements

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SIgA structure.

(A) Schematic representing ordered domains in the SIgA structure. The relative position of each SIgA component is approximated based on the structure. Chain IDs and corresponding C_H domains and Fcs are labeled along with SC domains (1-5). Each SIgA component is depicted in a unique color; J chain (JC), magenta; secretory component (SC), teal; heavy chains A, dark green, B, light green, C, dark blue, and D, light blue, respectively. (B) Cartoon representation (with semi-transparent molecular surface) of the SIgA structure shown in four orientations and colored as in (A). C_H and SC domains (D1-D5) are labeled and PNGS are shown as orange spheres. The bend and tilt between the two Fcs are indicated with a line and the angle measured in the structure; the concave and convex sides are labeled, along with the front face and the back face.

Figure 2—figure supplement 1

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SIgA cryoEM data collection and CryoSparc processing pipeline.

(A) Schematic summary of the data processing pipeline of SIgA reconstruction in cryoSparc2. (B) Representative micrograph (scale bar – 50 nm) and (C) 2D class averages. (D) Local resolution map of the final structure calculated in cryoSparc2 with FSC cut off at 0.143 threshold and prepared in ChimeraX; the scale bar was generated in Chimera. (E) FSC curves for the final reconstruction with reported resolution at FSC = 0.143 shown by the blue horizontal line. (F) Densities from the J and E chains contoured to 8σ and carved at 1.6 Å cutoff.

Figure 2—figure supplement 2

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SIgA cryoEM data collection and Relion processing pipeline.

(A) Schematic summary of the data processing pipeline of SIgA reconstruction in Relion. (B) Representative 2D class averages. (C) FSC curve for the final reconstruction with reported resolution at FSC = 0.143 shown by the horizontal dashed line. (D) Local resolution map of the final structure calculated in cryoSparc2 with FSC cut off at 0.143 threshold and prepared in ChimeraX; the scale bar was generated in Chimera. (E) Densities from the J and E chains contoured to 8σ and carved at 1.6 Å cutoff.

Figure 2—figure supplement 3

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SIgA cross-validation FSC curves.

The FSC curve generated between the final structure and CryoEM map including all data (FSC_map-model) is shown in black and indicates agreement between the map and final model. FSC_work (red) is the FSC curve generated between the final structure refined against half the data (half map 1) and half map 1. FSC_free (blue) is the FSC curve generated between the final structure refined against half map 1 and the other half of the data (half map 2), not used in refinement calculations. Close agreement between, FSC_work and FSC_free indicate that the model has not been overfit to the map.

Figure 2—figure supplement 4

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SIgA glycosylation.

Cartoon representations of SIgA structure with ordered glycosylation shown as sticks. The structure is shown in four orientations and colored as in Figure 2.

Figure 3

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Fc dimer interface and JC structure.

(A) Schematic depicting the topology that arises from JC and its interactions with four Tps and colored as in Figure 2. JC regions (JC_W1, JC_W2, JC_core, and N- and C termini) are labeled along with Tp_A-D and their location relative to the front face or back face of SIgA; disulfide bonds are indicated in yellow and the residue boundaries of each β-strand are labeled. (B) SIgA molecular surface representation (SC removed) colored as in Figure 2 and indicating the location of JC_W1, JC_W2 and Tps relative to each HC_A-D in two SIgA orientations. (C) Cartoon representation of JC and Tps complex (same region as shown in panel A) with the N- and C termini of each Tp shown as blue and red spheres, respectively. Regions surrounding the three C-terminal residues of Tp are boxed, Tp_A (green box), and Tp_C and Tp_D (blue boxes), and enlarged. Enlargements show Tp carboxy termini (COOH) and side chain sticks involved in interactions with adjacent Tps, JC, C_H3 domains, and SC. The three C-terminal residues of Tp_B are disordered and not shown. (D) Topology diagram and sequence (top) and structure (bottom) detailing interactions between JC_W2 and C_H3_A (left), JC_W1 and C_H3_C (center), and JC_W1 and SC (right) and colored as in A-C. JC residues interacting with C_H3 or SC are indicated in bold and numbered in the topology diagram (top) and shown as sticks on a cartoon representation (bottom); C_H3 and SC domains are shown as molecular surface representations with interfacing residues shown as sticks. Negatively charged atoms in C_H3 that form contacts are shown as a red surface.

Figure 4

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SC Structure.

(A) Cartoon representations of an unliganded mouse SC homology model based on unliganded human SC crystal structure (PDB code 5D4K; left) and the dIgA-bound mouse SC (right). The dIgA is shown as a molecular surface representation; SC domains D1-D5 are show in color gradient from cyan (D1) to pale green (D5), with CDR loops colored gray; PNGS are shown as orange spheres. Regions where SC interfaces with dIgA and other SC domains are boxed. (**B-D**) Boxed, enlarged views of stabilizing interfaces between SC Fc, JC and other SC domains; colored as in panel A. (B) SC D1 interface with C_H3_A, C_H3_B, Tp_C, Tp_D. (C) SC D1-D3 interface. (D) SC D4-D5 interface with JC and C_H2_C. In all panels conserved, interfacing residues are shown as sticks and hydrogen bonds and salt bridges are show as black dashes.

Figure 5 with 2 supplements

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dIgA structure.

(A) Cartoon representation (with semi-transparent molecular surface) of the unliganded dIgA structure shown in two orientations and colored as in Figure 2. The bend and tilt between the two Fcs is indicated with a line and the angle measured in the structure; the concave and convex sides are labeled, along with the front face and the back face and C_H domains. PNGS are shown as orange spheres. (B) Molecular surface representation of SIgA (gray; SC removed) aligned to dIgA (green) on the JC from each complex structure. (C) CryoEM maps and structures for SIgA (right) and dIgA (left) detailing the region surrounding Tp_D. (D) CryoEM maps and structures for SIgA (right) and dIgA (left) detailing the region surrounding the JC C-terminus.

Figure 5—figure supplement 1

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dIgA cryoEM data collection and processing strategy.

(A) Schematic summary of the data processing pipeline of dIgA reconstruction in cryoSparc2. (B) Representative micrograph (scale bar – 50 nm) and (C) 2D class averages. (D) Local resolution map of the final structure calculated in cryoSparc2 with FSC cut off at 0.143 threshold. (E) FSC curves for the final reconstruction with reported resolution at FSC = 0.143 shown by the blue horizontal line. (F) Densities from the J chain contoured to 8σ and carved at 1.9 Å cutoff.

Figure 5—figure supplement 2

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dIgA cross-validation FSC curves.

The FSC curve generated between the final structure and CryoEM map including all data (FSC_map-model) is shown in black and indicates agreement between the map and final model. FSC_work (red) is the FSC curve generated between the final structure refined against half the data (half map 1) and half map 1. FSC_free (blue) is the FSC curve generated between the final structure refined against half map 1 and the other half of the data (half map 2), not used in refinement calculations. Close agreement between, FSC_work and FSC_free indicate that the model has not been overfit to the map.

Figure 6 with 1 supplement

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Modeling of Fabs and CDRs on SIgA.

(A) SIgA structure shown with four Fabs, each modeled in a single position as well as all possible positions and shown in multiple orientations. Complex components are colored according to the key (center). (B) The number of positions sampled by Fab CDRs were quantified by measuring the angle (ϕ) between the Fab vector and the y-axis, a vector parallel to the FcAB plane and passing through the center of mass of Fc and JC. The frequency (normalized count) of each angle is shown as a histogram.

Figure 6—figure supplement 1

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Modeling strategy for the computational search of SIgA Fab positions.

(Left) SIgA structure shown with four modeled Fabs in one orientation. (Right) Schematic showing how Fab positions were modeled by mapping a vector, spanning from the N-terminal residue in the C_H2 domain (pivot) to the center of mass (C.O.M) of the CDRs (yellow), onto a Fibonacci spherical lattice (FSL) with one thousand points and γ angle intervals of 45°.

Figure 7

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Model for the formation, transport,and function of SIgA.

Schematic summary depicting the unliganded SC structure (pdb code 5D4K) as pIgR bound to basolateral surface of an epithelial cell in its closed conformation and recognizing bent dIgA from the lamina propria. The pIgR binding to dIgA triggers a conformation change that repositions its domains to facilitate numerous stabilizing contacts with dIgA. The dIgA-pIgR complex transcytoses to the apical membrane where the pIgR is proteolytically cleaved, releasing SIgA into the mucosa. In the mucosa, SIgA Fabs (shown in all possible modeled positions) are directed toward the concave side of the antibody ‘looking’ for potential antigens while its Fc-receptor-binding regions are exposed on the convex side and accessible to potential host or microbial receptors. SC domains are also partially exposed; the D2 domain is almost completely accessible, protruding out of the SIgA where it may bind host and bacterial factors. Upon encountering antigen, SIgA Fabs bind, promoting antigen coating, agglutination, or enchained growth.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Other	IGHA_MOUSE	UniProtKB RRID:SCR_004426	UniProtKB: P01878	Amino acid sequence of Mus musculus IgA heavy chain constant regions
Other	LAC1_MOUSE	UniProtKB RRID:SCR_004426	UniProtKB: P01843	Amino acid sequence of Mus musculus lambda-1 light chain constant region
Other	IGJ_MOUSE	UniProtKB RRID:SCR_004426	UniProtKB: P01592	Amino acid sequence of Mus musculus joining chain
Other	PIGR_MOUSE	UniProtKB RRID:SCR_004426	UniProtKB: O70570	Amino acid sequence of Mus musculus polymeric Ig Receptor
Other	STA121	Moor et al., 2017		Variable region of human IgA2 antibody STA121
Cell line (Homo sapiens)	HEK Expi293F	Thermo Fisher RRID:SCR_008452	Cat#: A14635 RRID:CVCL_D615
Recombinant DNA reagent	pD2610-v1	ATUM	Cat#: pD2610-v1-03	Mammalian expression plasmid
Transfected construct (Homo sapiens)	STA121 IgA Heavy chain (HC) in pD2610v1	Materials and methods of this paper		Construct encoding STA121 heavy chain (HC) variable region fused to Mus musculus IgA HC constant domains
Transfected construct (Homo sapiens)	STA121 IgA light chain (LC) lambda in pD2610v1	Materials and methods of this paper		Construct encoding STA121 light chain (LC) variable region fused to Mus musculus lambda LC constant domain
Transfected construct (Homo sapiens)	Mouse Secretory component (SC) in pD2610v1	Materials and methods of this paper		Construct encoding residues 1–567 of Mus musculus pIgR (a.k.a. secretory component; SC)
Transfected construct (Homo sapiens)	Mouse joining chain (JC) in pD2610v1	Materials and methods of this paper		Construct encoding Mus musculus joining chain (JC)
Peptide, recombinant protein	STA121 Secretory IgA	Materials and methods of this paper		Protein complex produced from transfected constructs and containing: STA121 HC, STA121 LC, JC, SC
Peptide, recombinant protein	STA121 dimeric IgA	Materials and methods of this paper		Protein complex produced from transfected constructs and containing: STA121 HC, STA121 LC, JC
Other	CaptureSelect LC-lambda (Mouse) Affinity Matrix	Thermo Fisher RRID:SCR_008452	Cat#: 194323005	Affinity matrix for protein purification
Other	Superose 6 Increase 10/300 GL	GE Healthcare Life Sciences RRID:SCR_000004	Cat#: 29091596	Size exclusion column for protein purification
Software, algorithm	Rosetta CryoEM refinement package	Wang et al., 2016	RRID:SCR_015701
Software, algorithm	Phenix	Afonine et al., 2018a; Afonine et al., 2018b	Phenix RRID:SCR_014224 Phenix.refine RRID:SCR_016736
Software, algorithm	Pymol Molecular Graphics System	Schrodinger LLC RRID:SCR_014879	RRID:SCR_000305
Software, algorithm	RELION-3	Scheres, 2012; Zivanov et al., 2018	RRID:SCR_016274
Software, algorithm	UCSF Chimera	Pettersen et al., 2004; Yang et al., 2012	RRID:SCR_004097
Software, algorithm	UCSF ChimeraX	Goddard et al., 2018	RRID:SCR_015872
Software, algorithm	cryoSPARC v.2	Punjani et al., 2017	RRID:SCR_016501
Software, algorithm	VMD	Humphrey et al., 1996	RRID:SCR_001820