Common use of Structural Model Clause in Contracts

Structural Model. The bundle designability landscape showed a single maximum “hotspot” at (ΔZ, ΔΦ) = (21.5 Å, −33.8°) (Fig. 1C) corresponding to bundle placement in which each pair of helices was offset in both translation and rotation to accommo- date the gentle left-handed supertwist of the bundles (SI Ap- pendix, Fig. S2A). Inspection of the designability landscapes of the four backbone connections revealed the positions of their hotspots had some degree of similarity (Fig. 1B and SI Appendix, Fig. S2B), yet they also differed due to underlying differences between bundle geometries (SI Appendix, Figs. S2–S5). Analo- gous designability searches considering each adjacent pair of helices in the bundle (i.e., helices 1 & 2, 2 & 3, 3 & 4, and 4 & 1), showed the same trends (SI Appendix, Fig. S3). Structural matches occurred when the translational and rotational offset between helical fragments were compatible with polypeptide linkers that adopted designable helix geometries. Moreover, the flexibility in those geometries allowed simultaneous structural matches to be realized in the four helices in the bundle. Downloaded by guest on October 15, 2021 In particular, the coordinates of the maxima in the bundle designability plot (Fig. 1C) identified the most favorable set of linker geometries (indicated by black dots in SI Appendix, Fig. S2B). The top structural matches to the disjointed helices (SI Appendix, Fig. S2C) all displayed helical geometry, with distor- tion from ideality in two cases (SI Appendix, Fig. S5). Helices 1–3 were best connected with a two-residue helical linker, whereas the structural matches for helix four consisted of five-residue and six-residue (SI Appendix, Fig. S2C, white, and SI Appendix, Fig. S4) linkers. To construct the final backbone structure, the four helical backbone fragments were connected into a single chain by incor- porating the N and C termini plus the loop from the DF structure (Fig. 1D, colored blue), and the loops and helical regions com- prising the folded core from the porphyrin-binding structure (Fig. 1D, colored yellow). Sequence design was restricted to the helical segments where the distinct bundles were connected, at residue positions that do not have side chains within the first and second shell of the dimetal-binding and porphyrin-binding sites. Backrub within Rosetta was used to sample small structural changes around the connections in conjunction with alternating loops of fixed-backbone sequence design and backbone/sidechain minimization (SI Appendix). The results from Rosetta were com- pared with sequence information from the MASTER searches (SI Appendix, Fig. S5), and these data sources along with visual in- spection of the model were used to finalize the primary structure. Structural Characterization of DFP1. DFP1 was cloned and expressed as described in Materials and Methods. For spectroscopic and structural characterization, we reconstituted the protein with ZnP in the porphyrin-binding site. Zn2+ was introduced into the dimetal site as a redox-inactive stable mimic of ferrous ions, as in previous studies of DF proteins. Spectral titration with ZnP demonstrated that the protein bound tightly to the cofactor. Time-resolved transient absorption spectroscopic data acquired for ZnP-DFP1 (SI Appendix, Fig. S6A) evinces spectral features and excited-state dynamics characteristic of the benchmark ZnP-PS1 holoprotein (41). Identical experiments carried out with di-Zn2+-ZnP-DFP1 (SI Appendix, Fig. S6B) indicate that Zn2+ occupancy of protein dimetal binding site does not perturb the excited-state relaxation dynamics of the ZnP chromophore or its characteristic electroni- cally excited singlet (S1)- and triplet (T1)-state absorptions. Because S1→T1 intersystem crossing rate constants of electronically excited porphyrins are known to be sensitive to both macrocycle structure and the local environment (50), these experiments demonstrate that the ZnP-binding site in PS1 is faithfully reproduced in DFP1. The structure of the holoprotein was solved by molecular re- placement to 3.5-Å resolution (Fig. 2A). At this resolution, the metal ions are very well resolved, and the density is sufficiently clear, as well as the OMIT maps, to allow placement of the por- phyrin macrocycle and the zinc ions (SI Appendix, Figs. S7 and S8). The closest distance between a porphyrin carbon and a Zn2+ ion of the dimetal cofactor is 12 Å. Furthermore, we investigated whether the individual domains of DFP1 preserved the structural features of the starting single-domain proteins. Indeed, we ob- served an excellent agreement considering individually the two domains: 0.74 Å rmsd for superposition of the ZnP domain versus PS1 and 0.52 Å for the dimetal binding domain versus DF1 (SI Appendix, Fig. S9). Moreover, the superposition of the 120 resi- dues comprising the cofactor binding sites gives an excellent fit to the design model with an rmsd = 0.8 Å (Fig. 2 B and C). At this resolution, it was not possible to detect deviations from ideal hydrogen-bonded geometries in the helices, but each connecting helix showed continuous density with B-factors within the range seen for the main chain atoms in other portions of the protein. Design and Solution Characterization of Catalytically Active DFP3. DFP1 was designed for maximal thermodynamic stability, and its interior is well-packed with apolar sidechains throughout the bundle. The resulting tight and uniform packing provided high stability, but did not leave room for organic substrates to access the dimetal-binding site. Therefore, the four interacting Leu and Ala residues located just above the dimetal site were substituted to Gly residues (Fig. 3 A and B, respectively), resulting in a deeply invaginated substrate access cavity. The resulting four-site mutant with Gly substitutions at positions 10, 14, 71, and 74 Xxxxx et al. PNAS Latest Articles | 3 of 8

Structural Model. The bundle designability landscape showed a single maximum “hotspot” at (ΔZ, ΔΦ) = (21.5 Å, −33.8°) (Fig. 1C) corresponding to bundle placement in which each pair of helices was offset in both translation and rotation to accommo- date the gentle left-handed supertwist of the bundles (SI Ap- pendix, Fig. S2A). Inspection of the designability landscapes of the four backbone connections revealed the positions of their hotspots had some degree of similarity (Fig. 1B and SI Appendix, Fig. S2B), yet they also differed due to underlying differences between bundle geometries (SI Appendix, Figs. S2–S5). Analo- gous designability searches considering each adjacent pair of helices in the bundle (i.e., helices 1 & 2, 2 & 3, 3 & 4, and 4 & 1), showed the same trends (SI Appendix, Fig. S3). Structural matches occurred when the translational and rotational offset between helical fragments were compatible with polypeptide linkers that adopted designable helix geometries. Moreover, the flexibility in those geometries allowed simultaneous structural matches to be realized in the four helices in the bundle. Downloaded by guest on October 15December 30, 2021 2020 In particular, the coordinates of the maxima in the bundle designability plot (Fig. 1C) identified the most favorable set of linker geometries (indicated by black dots in SI Appendix, Fig. S2B). The top structural matches to the disjointed helices (SI Appendix, Fig. S2C) all displayed helical geometry, with distor- tion from ideality in two cases (SI Appendix, Fig. S5). Helices 1–3 were best connected with a two-residue helical linker, whereas the structural matches for helix four consisted of five-residue and six-residue (SI Appendix, Fig. S2C, white, and SI Appendix, Fig. S4) linkers. To construct the final backbone structure, the four helical backbone fragments were connected into a single chain by incor- porating the N and C termini plus the loop from the DF structure (Fig. 1D, colored blue), and the loops and helical regions com- prising the folded core from the porphyrin-binding structure (Fig. 1D, colored yellow). Sequence design was restricted to the helical segments where the distinct bundles were connected, at residue positions that do not have side chains within the first and second shell of the dimetal-binding and porphyrin-binding sites. Backrub within Rosetta was used to sample small structural changes around the connections in conjunction with alternating loops of fixed-backbone sequence design and backbone/sidechain minimization (SI Appendix). The results from Rosetta were com- pared with sequence information from the MASTER searches (SI Appendix, Fig. S5), and these data sources along with visual in- spection of the model were used to finalize the primary structure. Structural Characterization of DFP1. DFP1 was cloned and expressed as described in Materials and Methods. For spectroscopic and structural characterization, we reconstituted the protein with ZnP in the porphyrin-binding site. Zn2+ was introduced into the dimetal site as a redox-inactive stable mimic of ferrous ions, as in previous studies of DF proteins. Spectral titration with ZnP demonstrated that the protein bound tightly to the cofactor. Time-resolved transient absorption spectroscopic data acquired for ZnP-DFP1 (SI Appendix, Fig. S6A) evinces spectral features and excited-state dynamics characteristic of the benchmark ZnP-PS1 holoprotein (41). Identical experiments carried out with di-Zn2+-ZnP-DFP1 (SI Appendix, Fig. S6B) indicate that Zn2+ occupancy of protein dimetal binding site does not perturb the excited-state relaxation dynamics of the ZnP chromophore or its characteristic electroni- cally excited singlet (S1)- and triplet (T1)-state absorptions. Because S1→T1 intersystem crossing rate constants of electronically excited porphyrins are known to be sensitive to both macrocycle structure and the local environment (50), these experiments demonstrate that the ZnP-binding site in PS1 is faithfully reproduced in DFP1. The structure of the holoprotein was solved by molecular re- placement to 3.5-Å resolution (Fig. 2A). At this resolution, the metal ions are very well resolved, and the density is sufficiently clear, as well as the OMIT maps, to allow placement of the por- phyrin macrocycle and the zinc ions (SI Appendix, Figs. S7 and S8). The closest distance between a porphyrin carbon and a Zn2+ ion of the dimetal cofactor is 12 Å. Furthermore, we investigated whether the individual domains of DFP1 preserved the structural features of the starting single-domain proteins. Indeed, we ob- served an excellent agreement considering individually the two domains: 0.74 Å rmsd for superposition of the ZnP domain versus PS1 and 0.52 Å for the dimetal binding domain versus DF1 (SI Appendix, Fig. S9). Moreover, the superposition of the 120 resi- dues comprising the cofactor binding sites gives an excellent fit to the design model with an rmsd = 0.8 Å (Fig. 2 B and C). At this resolution, it was not possible to detect deviations from ideal hydrogen-bonded geometries in the helices, but each connecting helix showed continuous density with B-factors within the range seen for the main chain atoms in other portions of the protein. Design and Solution Characterization of Catalytically Active DFP3. DFP1 was designed for maximal thermodynamic stability, and its interior is well-packed with apolar sidechains throughout the bundle. The resulting tight and uniform packing provided high stability, but did not leave room for organic substrates to access the dimetal-binding site. Therefore, the four interacting Leu and Ala residues located just above the dimetal site were substituted to Gly residues (Fig. 3 A and B, respectively), resulting in a deeply invaginated substrate access cavity. The resulting four-site mutant with Gly substitutions at positions 10, 14, 71, and 74 33248 | xxx.xxxx.xxx/xxx/xxx/00.0000/xxxx.0000000000 Xxxxx et al. PNAS Latest Articles | 3 of 8CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Structural Model. The bundle designability landscape showed a single maximum “hotspot” at (ΔZ, ΔΦ) = (21.5 Å, −33.8°) (Fig. 1C) corresponding to bundle placement in which each pair of helices was offset in both translation and rotation to accommo- date the gentle left-handed supertwist of the bundles (SI Ap- pendix, Fig. S2A). Inspection of the designability landscapes of the four backbone connections revealed the positions of their hotspots had some degree of similarity (Fig. 1B and SI Appendix, Fig. S2B), yet they also differed due to underlying differences between bundle geometries (SI Appendix, Figs. S2–S5). Analo- gous designability searches considering each adjacent pair of helices in the bundle (i.e., helices 1 & 2, 2 & 3, 3 & 4, and 4 & 1), showed the same trends (SI Appendix, Fig. S3). Structural matches occurred when the translational and rotational offset between helical fragments were compatible with polypeptide linkers that adopted designable helix geometries. Moreover, the flexibility in those geometries allowed simultaneous structural matches to be realized in the four helices in the bundle. Downloaded by guest on October 15September 8, 2021 In particular, the coordinates of the maxima in the bundle designability plot (Fig. 1C) identified the most favorable set of linker geometries (indicated by black dots in SI Appendix, Fig. S2B). The top structural matches to the disjointed helices (SI Appendix, Fig. S2C) all displayed helical geometry, with distor- tion from ideality in two cases (SI Appendix, Fig. S5). Helices 1–3 were best connected with a two-residue helical linker, whereas the structural matches for helix four consisted of five-residue and six-residue (SI Appendix, Fig. S2C, white, and SI Appendix, Fig. S4) linkers. To construct the final backbone structure, the four helical backbone fragments were connected into a single chain by incor- porating the N and C termini plus the loop from the DF structure (Fig. 1D, colored blue), and the loops and helical regions com- prising the folded core from the porphyrin-binding structure (Fig. 1D, colored yellow). Sequence design was restricted to the helical segments where the distinct bundles were connected, at residue positions that do not have side chains within the first and second shell of the dimetal-binding and porphyrin-binding sites. Backrub within Rosetta was used to sample small structural changes around the connections in conjunction with alternating loops of fixed-backbone sequence design and backbone/sidechain minimization (SI Appendix). The results from Rosetta were com- pared with sequence information from the MASTER searches (SI Appendix, Fig. S5), and these data sources along with visual in- spection of the model were used to finalize the primary structure. Structural Characterization of DFP1. DFP1 was cloned and expressed as described in Materials and Methods. For spectroscopic and structural characterization, we reconstituted the protein with ZnP in the porphyrin-binding site. Zn2+ was introduced into the dimetal site as a redox-inactive stable mimic of ferrous ions, as in previous studies of DF proteins. Spectral titration with ZnP demonstrated that the protein bound tightly to the cofactor. Time-resolved transient absorption spectroscopic data acquired for ZnP-DFP1 (SI Appendix, Fig. S6A) evinces spectral features and excited-state dynamics characteristic of the benchmark ZnP-PS1 holoprotein (41). Identical experiments carried out with di-Zn2+-ZnP-DFP1 (SI Appendix, Fig. S6B) indicate that Zn2+ occupancy of protein dimetal binding site does not perturb the excited-state relaxation dynamics of the ZnP chromophore or its characteristic electroni- cally excited singlet (S1)- and triplet (T1)-state absorptions. Because S1→T1 intersystem crossing rate constants of electronically excited porphyrins are known to be sensitive to both macrocycle structure and the local environment (50), these experiments demonstrate that the ZnP-binding site in PS1 is faithfully reproduced in DFP1. The structure of the holoprotein was solved by molecular re- placement to 3.5-Å resolution (Fig. 2A). At this resolution, the metal ions are very well resolved, and the density is sufficiently clear, as well as the OMIT maps, to allow placement of the por- phyrin macrocycle and the zinc ions (SI Appendix, Figs. S7 and S8). The closest distance between a porphyrin carbon and a Zn2+ ion of the dimetal cofactor is 12 Å. Furthermore, we investigated whether the individual domains of DFP1 preserved the structural features of the starting single-domain proteins. Indeed, we ob- served an excellent agreement considering individually the two domains: 0.74 Å rmsd for superposition of the ZnP domain versus PS1 and 0.52 Å for the dimetal binding domain versus DF1 (SI Appendix, Fig. S9). Moreover, the superposition of the 120 resi- dues comprising the cofactor binding sites gives an excellent fit to the design model with an rmsd = 0.8 Å (Fig. 2 B and C). At this resolution, it was not possible to detect deviations from ideal hydrogen-bonded geometries in the helices, but each connecting helix showed continuous density with B-factors within the range seen for the main chain atoms in other portions of the protein. Design and Solution Characterization of Catalytically Active DFP3. DFP1 was designed for maximal thermodynamic stability, and its interior is well-packed with apolar sidechains throughout the bundle. The resulting tight and uniform packing provided high stability, but did not leave room for organic substrates to access the dimetal-binding site. Therefore, the four interacting Leu and Ala residues located just above the dimetal site were substituted to Gly residues (Fig. 3 A and B, respectively), resulting in a deeply invaginated substrate access cavity. The resulting four-site mutant with Gly substitutions at positions 10, 14, 71, and 74 33248 | xxx.xxxx.xxx/xxx/xxx/00.0000/xxxx.0000000000 Xxxxx et al. PNAS Latest Articles | 3 of 8CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Structural Model. The bundle designability landscape showed a single maximum “hotspot” at (ΔZ, ΔΦ) = (21.5 Å, −33.8°) (Fig. 1C) corresponding to bundle placement in which each pair of helices was offset in both translation and rotation to accommo- date the gentle left-handed supertwist of the bundles (SI Ap- pendix, Fig. S2A). Inspection of the designability landscapes of the four backbone connections revealed the positions of their hotspots had some degree of similarity (Fig. 1B and SI Appendix, Fig. S2B), yet they also differed due to underlying differences between bundle geometries (SI Appendix, Figs. S2–S5). Analo- gous designability searches considering each adjacent pair of helices in the bundle (i.e., helices 1 & 2, 2 & 3, 3 & 4, and 4 & 1), showed the same trends (SI Appendix, Fig. S3). Structural matches occurred when the translational and rotational offset between helical fragments were compatible with polypeptide linkers that adopted designable helix geometries. Moreover, the flexibility in those geometries allowed simultaneous structural matches to be realized in the four helices in the bundle. Downloaded by guest on October 15April 27, 2021 In particular, the coordinates of the maxima in the bundle designability plot (Fig. 1C) identified the most favorable set of linker geometries (indicated by black dots in SI Appendix, Fig. S2B). The top structural matches to the disjointed helices (SI Appendix, Fig. S2C) all displayed helical geometry, with distor- tion from ideality in two cases (SI Appendix, Fig. S5). Helices 1–3 were best connected with a two-residue helical linker, whereas the structural matches for helix four consisted of five-residue and six-residue (SI Appendix, Fig. S2C, white, and SI Appendix, Fig. S4) linkers. To construct the final backbone structure, the four helical backbone fragments were connected into a single chain by incor- porating the N and C termini plus the loop from the DF structure (Fig. 1D, colored blue), and the loops and helical regions com- prising the folded core from the porphyrin-binding structure (Fig. 1D, colored yellow). Sequence design was restricted to the helical segments where the distinct bundles were connected, at residue positions that do not have side chains within the first and second shell of the dimetal-binding and porphyrin-binding sites. Backrub within Rosetta was used to sample small structural changes around the connections in conjunction with alternating loops of fixed-backbone sequence design and backbone/sidechain minimization (SI Appendix). The results from Rosetta were com- pared with sequence information from the MASTER searches (SI Appendix, Fig. S5), and these data sources along with visual in- spection of the model were used to finalize the primary structure. Structural Characterization of DFP1. DFP1 was cloned and expressed as described in Materials and Methods. For spectroscopic and structural characterization, we reconstituted the protein with ZnP in the porphyrin-binding site. Zn2+ was introduced into the dimetal site as a redox-inactive stable mimic of ferrous ions, as in previous studies of DF proteins. Spectral titration with ZnP demonstrated that the protein bound tightly to the cofactor. Time-resolved transient absorption spectroscopic data acquired for ZnP-DFP1 (SI Appendix, Fig. S6A) evinces spectral features and excited-state dynamics characteristic of the benchmark ZnP-PS1 holoprotein (41). Identical experiments carried out with di-Zn2+-ZnP-DFP1 (SI Appendix, Fig. S6B) indicate that Zn2+ occupancy of protein dimetal binding site does not perturb the excited-state relaxation dynamics of the ZnP chromophore or its characteristic electroni- cally excited singlet (S1)- and triplet (T1)-state absorptions. Because S1→T1 intersystem crossing rate constants of electronically excited porphyrins are known to be sensitive to both macrocycle structure and the local environment (50), these experiments demonstrate that the ZnP-binding site in PS1 is faithfully reproduced in DFP1. The structure of the holoprotein was solved by molecular re- placement to 3.5-Å resolution (Fig. 2A). At this resolution, the metal ions are very well resolved, and the density is sufficiently clear, as well as the OMIT maps, to allow placement of the por- phyrin macrocycle and the zinc ions (SI Appendix, Figs. S7 and S8). The closest distance between a porphyrin carbon and a Zn2+ ion of the dimetal cofactor is 12 Å. Furthermore, we investigated whether the individual domains of DFP1 preserved the structural features of the starting single-domain proteins. Indeed, we ob- served an excellent agreement considering individually the two domains: 0.74 Å rmsd for superposition of the ZnP domain versus PS1 and 0.52 Å for the dimetal binding domain versus DF1 (SI Appendix, Fig. S9). Moreover, the superposition of the 120 resi- dues comprising the cofactor binding sites gives an excellent fit to the design model with an rmsd = 0.8 Å (Fig. 2 B and C). At this resolution, it was not possible to detect deviations from ideal hydrogen-bonded geometries in the helices, but each connecting helix showed continuous density with B-factors within the range seen for the main chain atoms in other portions of the protein. Design and Solution Characterization of Catalytically Active DFP3. DFP1 was designed for maximal thermodynamic stability, and its interior is well-packed with apolar sidechains throughout the bundle. The resulting tight and uniform packing provided high stability, but did not leave room for organic substrates to access the dimetal-binding site. Therefore, the four interacting Leu and Ala residues located just above the dimetal site were substituted to Gly residues (Fig. 3 A and B, respectively), resulting in a deeply invaginated substrate access cavity. The resulting four-site mutant with Gly substitutions at positions 10, 14, 71, and 74 33248 | xxx.xxxx.xxx/xxx/xxx/00.0000/xxxx.0000000000 Xxxxx et al. PNAS Latest Articles | 3 of 8CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Structural Model. The bundle designability landscape showed a single maximum “hotspot” at (ΔZ, ΔΦ) = (21.5 Å, −33.8°) (Fig. 1C) corresponding to bundle placement in which each pair of helices was offset in both translation and rotation to accommo- date the gentle left-handed supertwist of the bundles (SI Ap- pendix, Fig. S2A). Inspection of the designability landscapes of the four backbone connections revealed the positions of their hotspots had some degree of similarity (Fig. 1B and SI Appendix, Fig. S2B), yet they also differed due to underlying differences between bundle geometries (SI Appendix, Figs. S2–S5). Analo- gous designability searches considering each adjacent pair of helices in the bundle (i.e., helices 1 & 2, 2 & 3, 3 & 4, and 4 & 1), showed the same trends (SI Appendix, Fig. S3). Structural matches occurred when the translational and rotational offset between helical fragments were compatible with polypeptide linkers that adopted designable helix geometries. Moreover, the flexibility in those geometries allowed simultaneous structural matches to be realized in the four helices in the bundle. Downloaded by guest on October 1523, 2021 In particular, the coordinates of the maxima in the bundle designability plot (Fig. 1C) identified the most favorable set of linker geometries (indicated by black dots in SI Appendix, Fig. S2B). The top structural matches to the disjointed helices (SI Appendix, Fig. S2C) all displayed helical geometry, with distor- tion from ideality in two cases (SI Appendix, Fig. S5). Helices 1–3 were best connected with a two-residue helical linker, whereas the structural matches for helix four consisted of five-residue and six-residue (SI Appendix, Fig. S2C, white, and SI Appendix, Fig. S4) linkers. To construct the final backbone structure, the four helical backbone fragments were connected into a single chain by incor- porating the N and C termini plus the loop from the DF structure (Fig. 1D, colored blue), and the loops and helical regions com- prising the folded core from the porphyrin-binding structure (Fig. 1D, colored yellow). Sequence design was restricted to the helical segments where the distinct bundles were connected, at residue positions that do not have side chains within the first and second shell of the dimetal-binding and porphyrin-binding sites. Backrub within Rosetta was used to sample small structural changes around the connections in conjunction with alternating loops of fixed-backbone sequence design and backbone/sidechain minimization (SI Appendix). The results from Rosetta were com- pared with sequence information from the MASTER searches (SI Appendix, Fig. S5), and these data sources along with visual in- spection of the model were used to finalize the primary structure. Structural Characterization of DFP1. DFP1 was cloned and expressed as described in Materials and Methods. For spectroscopic and structural characterization, we reconstituted the protein with ZnP in the porphyrin-binding site. Zn2+ was introduced into the dimetal site as a redox-inactive stable mimic of ferrous ions, as in previous studies of DF proteins. Spectral titration with ZnP demonstrated that the protein bound tightly to the cofactor. Time-resolved transient absorption spectroscopic data acquired for ZnP-DFP1 (SI Appendix, Fig. S6A) evinces spectral features and excited-state dynamics characteristic of the benchmark ZnP-PS1 holoprotein (41). Identical experiments carried out with di-Zn2+-ZnP-DFP1 (SI Appendix, Fig. S6B) indicate that Zn2+ occupancy of protein dimetal binding site does not perturb the excited-state relaxation dynamics of the ZnP chromophore or its characteristic electroni- cally excited singlet (S1)- and triplet (T1)-state absorptions. Because S1→T1 intersystem crossing rate constants of electronically excited porphyrins are known to be sensitive to both macrocycle structure and the local environment (50), these experiments demonstrate that the ZnP-binding site in PS1 is faithfully reproduced in DFP1. The structure of the holoprotein was solved by molecular re- placement to 3.5-Å resolution (Fig. 2A). At this resolution, the metal ions are very well resolved, and the density is sufficiently clear, as well as the OMIT maps, to allow placement of the por- phyrin macrocycle and the zinc ions (SI Appendix, Figs. S7 and S8). The closest distance between a porphyrin carbon and a Zn2+ ion of the dimetal cofactor is 12 Å. Furthermore, we investigated whether the individual domains of DFP1 preserved the structural features of the starting single-domain proteins. Indeed, we ob- served an excellent agreement considering individually the two domains: 0.74 Å rmsd for superposition of the ZnP domain versus PS1 and 0.52 Å for the dimetal binding domain versus DF1 (SI Appendix, Fig. S9). Moreover, the superposition of the 120 resi- dues comprising the cofactor binding sites gives an excellent fit to the design model with an rmsd = 0.8 Å (Fig. 2 B and C). At this resolution, it was not possible to detect deviations from ideal hydrogen-bonded geometries in the helices, but each connecting helix showed continuous density with B-factors within the range seen for the main chain atoms in other portions of the protein. Design and Solution Characterization of Catalytically Active DFP3. DFP1 was designed for maximal thermodynamic stability, and its interior is well-packed with apolar sidechains throughout the bundle. The resulting tight and uniform packing provided high stability, but did not leave room for organic substrates to access the dimetal-binding site. Therefore, the four interacting Leu and Ala residues located just above the dimetal site were substituted to Gly residues (Fig. 3 A and B, respectively), resulting in a deeply invaginated substrate access cavity. The resulting four-site mutant with Gly substitutions at positions 10, 14, 71, and 74 33248 | xxx.xxxx.xxx/xxx/xxx/00.0000/xxxx.0000000000 Xxxxx et al. PNAS Latest Articles | 3 of 8CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY