An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37
- Department of Chemistry & Biochemistry, University of California, San Diego, United States
- Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, University of California, San Diego, United States
Figures
Figure 1 with 1 supplement
LL-37 binding and detoxification.
(A) LL-37 binding to His-tagged M1HB, M58N100, or M87N100 as determined by Ni2+-NTA agarose bead co-precipitation assay at 37°C. The last lane contains no M protein. Bound fractions were resolved by …
Figure 1—figure supplement 1
LL-37 binding.
LL-37 binding to His-tagged M1HB, M4N100, M5N100, M22N100, M28N100, M44N100, M77N100, or M89N100 as determined by a Ni2+-NTA agarose bead co-precipitation assay at 37°C. The last lane contains no M …
Figure 2 with 1 supplement
Structures of GCN4-M87/LL-37 complex and free GCN4-M87.
(A) Cartoon representation of the GCN4-M87/LL-37 complex. LL-37 is in magenta. The GCN4 portion of GCN4-M87 is in gray, and the M87 portion in blue for the chain that makes more contacts to LL-37 …
Figure 2—figure supplement 1
Structures of the GCN4-M87/LL-37 complex and free GCN4-M87.
(A) Electron density for GCN4-M87 from a simulated annealing σA-weighted 2mFo-DFc composite omit map contoured at 1.4σ. Part of the interface between M87 (left) and LL-37 (right) is shown. (B) …
Figure 3
M87/LL-37 interface.
LL-37 is in magenta, and the M87 α-helix that forms a greater number of contacts with LL-37 is in blue (M87α1) and the one that forms fewer contacts in cyan (M87α2). Shown are contacts formed with …
Figure 4 with 2 supplements
Evaluation of M87/LL-37 interactions.
(A) LL-37 binding by His-tagged, intact wild-type M87 or M87 Y81A/I84A, L88A/F91A, E85A, E85R, W92A, or W92R at 37°C as determined by Ni2+-NTA agarose bead co-precipitation. The last lane contains …
Figure 4—figure supplement 1
Structure and stability of M87 mutant proteins.
(A) Circular dichroism (CD) spectra of intact wild-type and mutant M87 proteins at 37°C. The data are an average of two independent measurements. (B) Melting curves of intact wild-type and mutant …
Figure 4—figure supplement 2
Self-limiting growth of the M87/LL-37 complex.
Molecular weights of intact M87 protein and LL-37 added together and incubated for 1 hr (black) or 4 hr (blue) were determined by size-exclusion chromatography coupled to multiangle light …
Figure 5
Conservation of the M87LL-37-binding motif.
(A) M87 amino acids that make hydrophobic contact to LL-37 are boxed in red and those that disrupt interaction with LL-37 through Arg substitution are in blue boxes, as are amino acids that are …
Figure 6 with 1 supplement
LL-37-binding motif and higher-order complex formation.
(A) Occurrence of ideal heptads (dark gray boxes; Ile, Leu, Met, Phe, Tyr, or Val at both a and d positions) and near-ideal heptads (light gray boxes; ideal amino acid at a position and Ala at d …
Figure 6—figure supplement 1
LL-37-binding motif.
Depiction as in Figure 6A. The first grouping contains M proteins that have the LL-37-binding motif identified in M87 protein (experimentally verified for M87, M25, M58, and M68, and predicted for …
Tables
Table 1
Crystallographic data collection and model refinement.
| GCN4-M87/LL-37 | GCN4-M87 | |
|---|---|---|
| Data collection | ||
| Wavelength (Å) | 0.979 | 0.979 |
| Resolution range (Å) | 45.02 – 2.10 (2.18 – 2.10)* | 43.48 – 2.45 (2.54 – 2.45) |
| Space group | P21 | P43212 |
| Cell dimensions | ||
| a, b, c (Å) | 50.2, 57.1, 71.5 | 97.2, 97.2, 40.7 |
| α, β, γ (°) | 90.0, 102.7, 90.0 | 90.0, 90.0, 90.0 |
| Total reflections | 134,344 (9365) | 91,495 (8197) |
| Unique reflections | 22,092 (1694) | 7580 (738) |
| Multiplicity | 6.1 (5.5) | 12.1 (11.1) |
| Completeness (%) | 94.51 (89.58) | 99.75 (100.00) |
| I/σ(I) | 8.50 (2.10) | 31.40 (0.78) |
| Wilson B-factor (Å2) | 26.42 | 55.23 |
| † | 0.128 (0.735) | 0.404 (4.911) |
| CC1/2‡ | 0.996 (0.878) | 0.987 (0.556) |
| Refinement | ||
| Resolution range (Å) | 45.02 – 2.10 (2.20 – 2.10) | 43.48 – 2.45 (2.80 – 2.45) |
| No. of reflections (work/test set) | 21,958/2426 | 7563/383 |
| § | 0.22/0.27 (0.27/0.34) | 0.26/0.27 (0.29/0.38) |
| No. of non-hydrogen atoms | 2673 | 1138 |
| Macromolecules | 2556 | 1126 |
| Ligands | 16 (ethylene glycol) | 2 (PO43-) |
| Solvent | 101 | 2 |
| r.m.s. deviations | ||
| Bonds (Å) | 0.003 | 0.007 |
| Angles (°) | 0.46 | 0.95 |
| Ramachandran plot | ||
| Favored (%) | 100.00 | 100.00 |
| Outliers (%) | 0.00 | 0.00 |
| Rotamer outliers (%) | 0.00 | 0.00 |
| Clashscore | 3.37 | 3.98 |
| Average B-factor (Å2) | 46.59 | 76.91 |
| Macromolecules | 46.80 | 75.60 |
| Ligands | 37.52 | 110.74 |
| Solvent | 42.81 | 64.27 |
| Number of TLS groups | 8 | 2 |
| PDB code | 7SAY | 7SAF |
-
Formulas for and are identical except 95% of the total number of reflections was used to calculate , whereas the remaining 5% of reflection was used to calculate .
-
*Values in parentheses are for the highest resolution shell.
-
†
-
‡CC1/2 is the Pearson correlation between two half datasets.
-
§
Additional files
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/77989/elife-77989-transrepform1-v1.docx
-
Source data 1
Source data for Figure 1, Figure 1—figure supplement 1, Figure 4, and Figure 5.
- https://cdn.elifesciences.org/articles/77989/elife-77989-data1-v1.zip
