The dimerization interface of APL1B-LRR.

<p>A: Surface maps of the two APL1B-LRR molecules in the asymmetric unit showing N-terminal linked dimerization of the domains. B: Hydrophobic residues on the amphipathic N-terminal α-helix are protected from the solvent by a second APL1B-LRR domain. The hydrophobic residues (Val45, Leu48, Leu50) on the first LRR that are also protected by this interaction are shown. C: Backbone β-sheet interactions between two parallel β-strands (Met28–Leu30 and Ile55–Ile58) of the two APL1B-LRR domains that mediate the N-terminal dimerization interface. Electrostatic interactions between Ser27–Gln54 and His29–Asp59 are also shown. Hydrogen bonds are shown as dark blue dashes. Crystal structures of (D) APL1B-LRR, (E) APL1C-LRR and (F) LRIM1-LRR looking down the solenoid from the N-terminus. The secondary structural elements that are similar between the N-terminus of APL1B (helix from second chain) and the convex face of LRR II of APL1C are highlighted in yellow. Individual LRRs are numbered.</p

SAXS modeling of TEP1*R1, LRIM1-LRR, APL1C-LRR and LRIM1/APL1C.

<p>Superposition of <i>ab initio</i> SAXS models and best single model generated by <i>ALLOSMOD-FOXS</i> for TEP1*R1, LRIM1/APL1C, APL1C-LRR and LRIM1-LRR SAXS data. (A) Bead model displayed as green surface, protein model by red/blue cartoon with CPK sticks for N-linked glycosylation. (B) Fit to experimental scattering curve for each of the static structural models shown above.</p

Data collection and refinement statistics for the crystal structure of APL1B-LRR.

<p>Data collection and refinement statistics for the crystal structure of APL1B-LRR.</p

Biophysical Analysis of <i>Anopheles gambiae</i> Leucine-Rich Repeat Proteins APL1A¹, APL1B and APL1C and Their Interaction with LRIM1

<div><p>Natural infection of <i>Anopheles gambiae</i> by malaria-causing <i>Plasmodium</i> parasites is significantly influenced by the <i>APL1</i> genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, <i>APL1A</i>, <i>APL1B</i> and <i>APL1C</i>. Multiple studies have reported the participation of <i>APL1A—C</i> in the immune response of <i>A</i>. <i>gambiae</i> to invasion by both rodent and human <i>Plasmodium</i> isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects <i>A</i>. <i>gambiae</i> from infection by binding the complement-like protein TEP1 to form a stable and active immune complex. Here we report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A¹ and APL1B form a stable complex with LRIM1. These results support a model in which APL1A¹, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect <i>A</i>. <i>gambiae</i> from a diverse array of pathogens.</p></div

Disulfide-linked heterodimerization between full-length LRIM1 and APL1A—C.

<p>A: Schematic diagram showing the constructs that were used for the coIP experiment: signal peptide (black), low-complexity region (white), LRR domains (blue). Coiled-coil (green) FLAG tag (magenta), 6×His tag (cyan), cysteines (red lines). B: CoIP of FLAG-tagged LRIM1 with 6×His-tagged LRIM1, APL1A¹, APL1A², APL1B and APL1C. Western blots were performed with α6×His/HRP (top panel) and αFLAG/HRP (bottom panel) to detect the 6×His-tagged proteins and coIPed FLAG-tagged LRIM1, respectively. C: Conditioned media containing 6×His-tagged LRIM1, LRIM1-C352S, APL1C, APL1B and APL1A² (intracellular) were collected and evaluated with reducing (+DTT) or non-reducing (-DTT) SDS-PAGE and α6×His/HRP Western blotting.</p

Parameters derived by primary analysis of SAXS data.

<p>Parameters derived by primary analysis of SAXS data.</p

Schematic diagram of the LRIM1 and APL1 proteins.

<p>Colored boxes: black, signal peptide; white, low-complexity PANGGL region; yellow, Cys-rich region; green, coiled-coil (CC) domain. Boxes represent the number of LRR repeats for each protein. Features: loop between CC domains, helix-loop-helix (HLH) region; red line, Cys residue.</p

Alignment of the N-termini of APL1B and APL1C.

<p>Conserved residues are highlighted in black. The secondary structural elements that are similar between the N-terminus of APL1B and the convex face of LRR II of APL1C are highlighted in yellow. The LRRs are numbered and shown in grey boxes for APL1B and APL1C. The secondary structure elements of APL1B (PDB ID 4XGO) and APL1C (PDB ID 3O6N) are shown as coils for α-helices and arrows for β-strands.</p

Molecular weight of LRIM1/APL1 LRR domains in solution.

<p>Molecular weight of LRIM1/APL1 LRR domains in solution.</p

Solution State of LRIM1 and APL1A—C LRR domains.

<p>Solution MW determination of the LRR domains of APL1A¹, APL1B, APL1C and LRIM1 by SEC. The retention volumes of molecular mass standards in kDa are indicated on the bottom panel. (Inset) Purity of each protein on SDS-PAGE, lanes: (1) APL1A¹, (2) APL1B, (3) APL1C and (4) LRIM1.</p