Volume 24, Issue 7, Pages 1192-1200 (July 2016) Molecular Mechanism for Fungal Cell Wall Recognition by Rice Chitin Receptor OsCEBiP  Simiao Liu, Jizong Wang, Zhifu Han, Xinqi Gong, Heqiao Zhang, Jijie Chai  Structure  Volume 24, Issue 7, Pages 1192-1200 (July 2016) DOI: 10.1016/j.str.2016.04.014 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Overall Structure of OsCEBiP-ECD in Chitin Binding Form (A) Quantification of chitin binding affinity of OsCEBiP-ECD by isothermal titration calorimetry (ITC). Raw data (upper panel) and integrated heat measurements (lower panel) from ITC of the chitin tetramer binding to OsCEBiP-ECD are shown. The binding constant (K), the calculated stoichiometry (N), and the dissociation constant (Kd) are indicated. (B) ITC of chitin octamer binding to OsCEBiP-ECD. (C) ITC of titration of chitin tetramer into OsCERK1-ECD. (D) ITC of titration of chitin octamer into OsCERK1-ECD. (E) Colored-coded domain architecture of full-length OsCEBiP. The same color scheme is used in structure figures. SP, signal peptide; LysM, lysine motif; CRD, cysteine-rich domain; TM, transmembrane domain. (F) Overall structure of OsCEBiP-ECD in complex with chitin tetramer in two different orientations. The bound chitin oligomer is labeled with NAG and shown as green sticks. The secondary-structure elements are labeled. (G) Structure superposition of the three LysMs in OsCEBiP ectodomain. The secondary-structure elements of LysM1 are labeled. The color codes are indicated. See also Figure S1. Structure 2016 24, 1192-1200DOI: (10.1016/j.str.2016.04.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Structural and Functional Analysis of the Novel Structural Fold of CRD (A) Overall structure of CRD. The cysteine residues forming the disulfide bridges are indicated. The disulfide bonds are colored yellow and marked by red numbers. (B) Detailed interaction of βA sheet from CRD with α2 helix from LysM1. The side chains from βA sheet and α2 helix are shown in gray and yellow, respectively. (C) ITC of chitin tetramer binding to OsCEBiP-ECDΔCRD. (D) ITC of chitin octamer binding to OsCEBiP-ECDΔCRD. (E) Overall structure of OsCEBiP-ECDΔCRD. The color codes are indicated. (F) Structural superposition of OsCEBiP-ECDΔCRD and chitin-bound OsCEBiP-ECD. The structure of OsCEBiP-ECDΔCRD (residues 29–223) was used as the template for alignment with that of chitin-bound OsCEBiP-ECD (residues 29–223) with a root-mean-square deviation of 0.83 Å. See also Figure S2. Structure 2016 24, 1192-1200DOI: (10.1016/j.str.2016.04.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Recognition Mechanism of NAG Oligomers by LysM-Containing Receptors (A) Chitin residues are labeled (NAG-1, -2, -3) and the omit electron density (Fo-Fc, 2.7σ) around the bound chitin oligomer is shown in blue mesh. LysM2 is shown in electrostatic surface and cartoon. (B) Detailed interactions between chitin tetramer and the OsCEBiP-ECD. The side chains from OsCEBiP-ECD are shown in yellow. (C) Mutations of amino acids critical for the interaction of OsCEBiP with chitin compromise the chitin tetramer (NAG)4 binding activity of OsCEBiP-ECD. The data summarized here represent the dissociation constants (Kd) derived from ITC experiments. The raw data are shown in Figure S1. (D) The detailed model of hydrogen bonds and hydrophobic interaction between three NAG oligomers and one LysM. Seven critical residues (R1–R7) can be defined and labeled. (E) (Top) Structure-based sequence alignment indicates the conserved position of R1–R7 in the secondary structures. R1 is before the conserved Asp (D) on loop L1. R2, R3 is the first and second residue of helix α1, respectively. R4 is the first residue of helix α2. R5 is slightly variable and is boxed in red. R6 is the conserved Ile (I) or Leu (L) on loop L2. R7 is insulated one residue backward of R6. (Bottom ) Sequence alignment of LysM2 in several LysMs-containing RLKs and RLPs can predict potential critical residues (labeled R1–R7) for NAG oligomer binding according to the rules described above. The residues boxed in red can be the other candidates for R5. Consensus and similar amino acid residues for all sequences are highlighted in red and shown in blue boxes, respectively. See also Figures S1–S3. Structure 2016 24, 1192-1200DOI: (10.1016/j.str.2016.04.014) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Structure-Based Models of Chitin-Induced Homodimerization of OsCEBiP-ECD (A) Structure alignment of LysM2 in OsCEBiP-ECD to the LysM in the (NAG)6-mediated NlpC/P60-LysM homodimer (yellow, PDB: 4UZ3). Color codes are indicated. (B) Modeled structure of hexachitin-induced OsCEBiP-ECD homodimer with HoDock methods. Color codes are indicated. (C) Detailed interaction of hexachitin and two OsCEBiP-ECD boxed in solid black and red in (B). The side chains are shown in yellow. The critical residues of OsCEBiP-ECD-1 are marked in black and those of OsCEBiP-ECD-2 are marked in red. (D) The structure details boxed by black dashed line in (B). It forms the interface between the two OsCEBiP and shows slight interaction. See also Figures S3 and S4. Structure 2016 24, 1192-1200DOI: (10.1016/j.str.2016.04.014) Copyright © 2016 Elsevier Ltd Terms and Conditions