Alexandre R. Gingras, Umakhanth Venkatraman Girija, Anthony H. Keeble, Roshni Panchal, Daniel A. Mitchell, Peter C.E. Moody, and Russell Wallis

Size: px

Start display at page:

Download "Alexandre R. Gingras, Umakhanth Venkatraman Girija, Anthony H. Keeble, Roshni Panchal, Daniel A. Mitchell, Peter C.E. Moody, and Russell Wallis"

Lynette Fletcher
5 years ago
Views:

1 Structure, 19 Supplemental Information Structural Basis of Mannan-Binding Lectin Recognition by Its Associated Serine Protease MASP-1: Implications for Complement Activation Alexandre R. Gingras, Umakhanth Venkatraman Girija, Anthony H. Keeble, Roshni Panchal, Daniel A. Mitchell, Peter C.E. Moody, and Russell Wallis Inventory of Supplemental Information Figure S1 links to Figure 2 and shows the sequence similarities at the protease-binding sites of MBLs, ficolins, C1q and CL-K1 Figure S2 links to Figure 3 and shows how the mode of MBL-MASP binding differs from a previous model and why binding is orientation specific. Figure S3 links to Figure 4 and shows sequence alignments of CUB domains of MASPs, C1r and C1s. It also shows the autoinhibited MASP in which a lysine residue from on CUB domain interacts with its binding partner, mimicking the natural MBL-MASP CUB interaction. Figure S4 links to Figure 5 and shows the complement and protein inhibition data together with structures of MASP bound to small inhibitors.

Sequence alignment of rat and human MBLs, ficolins, CL- K1 (an MBL-like protein that binds to MASP-1) and the

2 Supplemental Figures Figure S1, related to Figure 2: Sequence alignment of the collagenous regions of MBL, ficolins, C1q, and CL-K1. Sequence alignment of rat and human MBLs, ficolins, CL- K1 (an MBL-like protein that binds to MASP-1) and the three chains of human C1q. Symbols denote the conserved (*) residues in all molecules. Numbering is based on the sequence of rat MBL-A. Residues involved in MASP-1 CUB2 binding and equivalents are highlighted.

Figure S2, related to Figure 3. Crystal structure of MASP-1 CUB2 bound to the 27- residue MBL collagen-like peptide 1.

(Inset) A previous model of MBL-MASP interaction showing the same view of the CUB domain.

(B) In the CUB-collagen structure, His218 and Tyr225 occupy a groove in the collagen and form hydrogen bonds to the peptide backbone (Leu47 of the

This groove is lined by residues of the common MASP-binding motif (OGKLGP), so will be similar in MBLs, ficolins, CL-K1 and C1q.

3 Figure S2, related to Figure 3. Crystal structure of MASP-1 CUB2 bound to the 27- residue MBL collagen-like peptide 1. (A) The CUB2 domain (green) is located on the outside of the cone created by the four MBL subunits (left). The collagen is in white. (Inset) A previous model of MBL-MASP interaction showing the same view of the CUB domain. The MBL stem is inverted and makes different contacts with the MASP. (B) In the CUB-collagen structure, His218 and Tyr225 occupy a groove in the collagen and form hydrogen bonds to the peptide backbone (Leu47 of the leading strand and Gly45 of the trailing strand). This groove is lined by residues of the common MASP-binding motif (OGKLGP), so will be similar in MBLs, ficolins, CL-K1 and C1q. The adjacent groove is occupied by Ser265. (D) In MBL (modelled in the figure), the adjacent groove would be filled by Gln41 and Leu40 of the trailing chain (indicated by an apostrophe). Although the small serine side chain (Ser265) can still be accommodated in the binding orientation, these bulky residues would prevent binding to the collagen in the inverted orientation, through steric clashes with His218, Tyr225 and the other residues of loop L5.

Secondary structure elements are indicated and numbering is based on rat MASP-1. Symbols denote the degree of conservation: (*) identical, (:) conservative and (.) semi-conservative substitution.

4 Figure S3, related to Figure 4: Conserved mode of binding by Ca 2+ -dependent CUB domains. Sequence alignment of: (A) CUB2 domains from human (h) and rat proteases (r), and (B) the CUB2 and CUB1 domains of rat MASP-1. Secondary structure elements are indicated and numbering is based on rat MASP-1. Symbols denote the degree of conservation: (*) identical, (:) conservative and (.) semi-conservative substitution. CUB2 of C1s does not bind to C1q. His and Tyr residues (at positions 218 and 225 of MASP-1) are replaced by Ala and Leu (red), respectively. (C) and (D) CUB domain crystal form 3 with an inter-molecular interaction mimicking the MBL mode of binding. (C) Three CUB2 molecules in the asymmetric unit form a cloverleaf shape. (D) A close-up view shows a pseudo autoinhibited form where K189 from each molecule projects into the binding pocket of its adjacent partner, mimicking the collagen/cub interaction. One of the CUB molecules is

Structures of the lysine-binding pocket of MASP-1 CUB2; (A) in the free protein, (B) bound to the MBL collagen peptide, (C) bound to methylamine and (D) bound to ethylamine.

5 represented as an electrostatic potential surface to highlight the charge and surface complementarity. Figure S4, related to Figure 5: Inhibition of complement activation and MBL-MASP binding by small amines. Structures of the lysine-binding pocket of MASP-1 CUB2; (A) in the free protein, (B) bound to the MBL collagen peptide, (C) bound to methylamine and (D) bound to ethylamine. (E) Inhibition of lectin pathway-dependent complement activation by lysine measured by deposition of complement component C3c in mouse serum. Data are averages ± difference from duplicate measurements. The IC 50 value for lysine was 29.7 ± 0.5 mm from two separate experiments. (F) Inhibition of full-length MASP-1 binding to immobilized MBL (including S.E.) by surface plasmon resonance. Data are expressed as a fraction of binding in the absence of inhibitors. IC 50 values are 49 ± 9, 193 ± 39 and 228 ± 43

6 mm for lysine, ethylamine and methylamine, respectively, from three independent experiments.

Gene function at the level of traits Gene function at the molecular level

Gene expression Gene function at the level of traits Gene function at the molecular level Two levels tied together since the molecular level affects the structure and function of cells which determines