A Model for the Molecular Mechanism of an Engineered Light-Driven Protein Machine  Daniel Hoersch, Tanja Kortemme  Structure  Volume 24, Issue 4, Pages 576-584 (April 2016) DOI: 10.1016/j.str.2016.02.015 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 576-584DOI: (10.1016/j.str.2016.02.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Simulated Global Structural Changes of Engineered Light-Switchable Mm-cpn Variants in Response to Distance Constraints (A) Single-particle cryo-electron microscopy reconstructions of ABDM crosslinked Mm-cpn mutant ΔC/S199C/K87C (Hoersch et al., 2013) in its closed and open states (EMDB: EMD-5769, EMD-5770). ΔC denotes C140S-C237S-C286A-C359T-C393A-C470S-C484S. The dashed lines enclose two neighboring monomers of the 16-mer homo-oligomeric protein complex. (B) Backbone models of two neighboring Mm-cpn subunits in the closed and open states derived from cryo-electron microscopy (PDB: 3IYE, 3IYF). Bars connect the Cα atoms of the sequence positions 199/87 (pink), 202/132 (red), and 204/129 (green) used previously for site-specific crosslinking with ABDM (Hoersch et al., 2013). (C) Chemical structure of the crosslinker ABDM in the two isomerization states cis and trans (models created with the software Avogadro (Hanwell et al., 2012)). Yellow sticks represent the sulfur atoms of the crosslinked cysteine residues. (D) Structural response of the closed Mm-cpn ring (left panel) to an imposed distance constraint of 19 Å between the crosslinking sites during the in silico relaxation: Cα-Cα rmsd of the tenth percentile lowest-energy relaxed models to the closed-state starting structure plotted against their Rosetta energy score (Rosetta energy units, r.e.u.) for the crosslinking sites 199/87, 204/129, 202/132, and 66/385 (black dots). Blue dots indicate the rmsd for the low-energy models after relaxation with the same protocol but without a distance constraint. The histograms on the right panels depict the rmsd distribution for each simulation. The frequency is normalized so that the area under the curve equals 1. Structure 2016 24, 576-584DOI: (10.1016/j.str.2016.02.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Correlation with the Electron Density Map of the Open State (A) Correlation of a simulated electron density map for the low-energy models relaxed with a distance constraint (black dots) with the electron density map of the open state of Mm-cpn (EMDB: EMD-5770) plotted against their Rosetta energy score (r.e.u.). The dashed line at a correlation value of 0.915 represents the correlation of the open state electron density to the atomic model of the open state of a lid-less construct of Mm-cpn (PDB: 3IYF). Blue dots indicate the correlation for the low-energy models after relaxation with the same protocol but without a distance constraint. The histograms on the right show the distribution of the correlations for the low-energy models. The red curves correspond to a fit with a sum of two normal distributions with the parameters: center (amplitude) 199/87: 0.79(0.41), 0.88(0.60) 204/129: 0.78(0.24), 0.86(0.78); 202/132: 0.69(0.92), 0.81(0.09); 66/385: 0.75(0.92), 0.86(0.07). (B) Best overlapping models (colored by individual subunits) from (A), shown together with a contour map of the electron density of the open state (gray). Note that the electron density of the protruding α helix in the apical domain is missing due to the inherent flexibility of the open state (Hoersch et al., 2013; Zhang et al., 2010). Structure 2016 24, 576-584DOI: (10.1016/j.str.2016.02.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Lid Opening (A) Top view of the structure of the closed state of Mm-cpn (PDB: 3RUV). The dotted circle encloses the lid structure composed of protruding α helices of the apical domains of the subunits of each chaperonin ring. Residue I250 is shown in pink space fill. The pink dotted line connects I250 in opposing subunits. (B) Cα-Cα distances of I250 in opposing subunits in the tenth percentile lowest-energy models relaxed with a distance constraint (black dots). Blue dots indicate the corresponding distance for the low-energy models after relaxation with the same protocol but without a distance constraint. The dashed line indicates a distance of 23 Å used to bin the models into closed- and open-lid populations as shown in the panels on the right. The dotted lines on the right indicate the I250 Cα-Cα distance of the models with high overlap to the open-state electron density shown in Figure 2B. Structure 2016 24, 576-584DOI: (10.1016/j.str.2016.02.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Subdomain Rotation Angle and SASA of the Nucleotide (A) Subdomain composition and nucleotide-binding site of Mm-cpn (PDB: 3RUV). Depicted in yellow and cyan are the residues of the equatorial and intermediate domain, respectively, that are located within a 5 Å shell around the nucleotide. (B) Plot of the rotation angle between the equatorial and intermediate domains against Rosetta score for the low-energy models relaxed with a distance constraint (black dots). Blue dots indicate the rotation angle for the low-energy models after relaxation with the same protocol but without a distance constraint. The histograms on the right show the distribution of the rotation angle. The red curves correspond to a fit with a normal distribution. (C) Nucleotide SASA plotted against Rosetta score for the low-energy models relaxed with a distance constraint (black dots). Blue dots indicate the SASA for the low-energy models after relaxation with the same protocol but without a distance constraint. The histograms show the SASA distribution. The red curves correspond to a fit with a normal distribution. Structure 2016 24, 576-584DOI: (10.1016/j.str.2016.02.015) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Light-Induced Conformational Change for the Crosslinked Mm-cpn Mutant R66C-C140S-C237S-L258W-C286A-D385C-C393A-C470S-C484S (A) Location of the engineered crosslinking site. The red bar connects the Cα atoms of residues 66 and 385 in the closed state structure of the Mm-cpn monomer (PDB: 3RUV). The nucleotide is shown in space fill (magenta). (B) Crosslinking of R66C-C140S-C237S-L258W-C286A-D385C-C393A-C470S-C484S Mm-cpn with cis-ABDM (indicated by pink ∧ symbol) to generate crosslinked Mm-cpn, and visualization of the light-induced switching between the open and closed state of Mm-cpn using a non-denaturing PAGE gel. (C) Quantification of the fraction of open state present in the sample after each illumination step using either blue (450 nm, blue star) or UV (365 nm, cyan star) light from data shown in (B). Structure 2016 24, 576-584DOI: (10.1016/j.str.2016.02.015) Copyright © 2016 Elsevier Ltd Terms and Conditions