The structures of secretory and dimeric immunoglobulin A

  1. Sonya Kumar Bharathkar
  2. Benjamin W Parker
  3. Andrey G Malyutin
  4. Nandan Haloi
  5. Kathryn E Huey-Tubman
  6. Emad Tajkhorshid
  7. Beth M Stadtmueller  Is a corresponding author
  1. Department of Biochemistry, University of Illinois Urbana-Champaign, United States
  2. Division of Biology and Biological Engineering, California Institute of Technology, United States
  3. Beckman Institute, California Institute of Technology, United States
  4. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana–Champaign, United States
  5. NIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute for Advanced Science and Technology, United States
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
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 …

Figure 1—figure supplement 1
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 …

Figure 2 with 4 supplements
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 CH domains and Fcs …

Figure 2—figure supplement 1
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 …

Figure 2—figure supplement 2
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 …

Figure 2—figure supplement 3
SIgA cross-validation FSC curves.

The FSC curve generated between the final structure and CryoEM map including all data (FSCmap-model) is shown in black and indicates agreement between the map and final model. FSCwork (red) is the …

Figure 2—figure supplement 4
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.

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 (JCW1, JCW2, JCcore, and N- and C termini) are labeled along with …

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 …

Figure 5 with 2 supplements
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 …

Figure 5—figure supplement 1
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 …

Figure 5—figure supplement 2
dIgA cross-validation FSC curves.

The FSC curve generated between the final structure and CryoEM map including all data (FSCmap-model) is shown in black and indicates agreement between the map and final model. FSCwork (red) is the …

Figure 6 with 1 supplement
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 …

Figure 6—figure supplement 1
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 CH2 …

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 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
OtherIGHA_MOUSEUniProtKB
RRID:SCR_004426
UniProtKB: P01878Amino acid sequence of Mus musculus IgA heavy chain constant regions
OtherLAC1_MOUSEUniProtKB
RRID:SCR_004426
UniProtKB: P01843Amino acid sequence of Mus musculus lambda-1 light chain constant region
OtherIGJ_MOUSEUniProtKB
RRID:SCR_004426
UniProtKB: P01592Amino acid sequence of Mus musculus joining chain
OtherPIGR_MOUSEUniProtKB
RRID:SCR_004426
UniProtKB: O70570Amino acid sequence of Mus musculus polymeric Ig Receptor
OtherSTA121Moor et al., 2017Variable region of human IgA2 antibody STA121
Cell line (Homo sapiens)HEK Expi293FThermo Fisher
RRID:SCR_008452
Cat#: A14635
RRID:CVCL_D615
Recombinant DNA reagentpD2610-v1ATUMCat#: pD2610-v1-03Mammalian expression plasmid
Transfected construct (Homo sapiens)STA121 IgA Heavy chain (HC) in pD2610v1Materials and methods of this paperConstruct 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 pD2610v1Materials and methods of this paperConstruct encoding STA121 light chain (LC) variable region fused to Mus musculus lambda LC constant domain
Transfected construct
(Homo sapiens)
Mouse Secretory component (SC) in pD2610v1Materials and methods of this paperConstruct encoding residues 1–567 of Mus musculus pIgR (a.k.a. secretory component; SC)
Transfected construct
(Homo sapiens)
Mouse joining chain (JC) in pD2610v1Materials and methods of this paperConstruct encoding
Mus musculus joining chain (JC)
Peptide, recombinant proteinSTA121 Secretory IgAMaterials and methods of this paperProtein complex produced from transfected constructs and containing: STA121 HC, STA121 LC, JC, SC
Peptide, recombinant proteinSTA121 dimeric IgAMaterials and methods of this paperProtein complex produced from transfected constructs and containing: STA121 HC, STA121 LC, JC
OtherCaptureSelect LC-lambda (Mouse) Affinity MatrixThermo Fisher
RRID:SCR_008452
Cat#: 194323005Affinity matrix for protein purification
OtherSuperose 6 Increase 10/300 GLGE Healthcare Life Sciences
RRID:SCR_000004
Cat#: 29091596Size exclusion column for protein purification
Software, algorithmRosetta CryoEM refinement packageWang et al., 2016RRID:SCR_015701
Software, algorithmPhenixAfonine et al., 2018a; Afonine et al., 2018bPhenix RRID:SCR_014224
Phenix.refine RRID:SCR_016736
Software, algorithmPymol Molecular Graphics SystemSchrodinger LLC
RRID:SCR_014879
RRID:SCR_000305
Software, algorithmRELION-3Scheres, 2012; Zivanov et al., 2018RRID:SCR_016274
Software, algorithmUCSF ChimeraPettersen et al., 2004; Yang et al., 2012RRID:SCR_004097
Software, algorithmUCSF ChimeraXGoddard et al., 2018RRID:SCR_015872
Software, algorithmcryoSPARC v.2Punjani et al., 2017RRID:SCR_016501
Software, algorithmVMDHumphrey et al., 1996RRID:SCR_001820

Additional files

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