Volume 26, Issue 2, Pages 199-208.e3 (February 2018) Structural Mechanism for the Temperature-Dependent Activation of the Hyperthermophilic Pf2001 Esterase  Nathalia Varejão, Rafael A. De-Andrade, Rodrigo V. Almeida, Cristiane D. Anobom, Debora Foguel, David Reverter  Structure  Volume 26, Issue 2, Pages 199-208.e3 (February 2018) DOI: 10.1016/j.str.2017.12.004 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Thermal-Induced Dimerization of the Pf2001 Esterase Assessed by Size-Exclusion Chromatography and Crosslinking Experiment Followed by SDS-PAGE (A) Effects of temperature on esterase activity using 4-methylumbelliferyl heptanoate as substrate. The purified esterase was assayed at temperatures ranging from 50°C to 80°C, in 50  mM phosphate buffer (pH 7.0). Data values are mean ± SD of three technical replicates. (B) The purified monomer (2 mg/mL) was maintained at 6°C or incubated at 55°C for 15 min before injection into a Superdex 200 HiLoad 16/600 column kept at the desired temperature by using a water jacket system. Insets show the protein content of each elution peak by SDS-PAGE. (C) SDS-PAGE of Pf2001 esterase performed after covalent crosslinking at 6°C and 55°C. Samples (1 mg/mL) incubated at each temperature for 5 min were mixed with glutaraldehyde (0.05%) for 30 s and then stopped using 1.6 M Tris-HCl buffer as described in the STAR Methods. Proteins were then mixed with SDS loading buffer and resolved by SDS-PAGE. Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Monomeric and Dimeric Structures of Pf2001: Rearrangement of the Cap Subdomain between Monomer and Dimer (A) Ribbon representation of the monomeric P3121 crystal form of the Pf2001 esterase. Ser149 is labeled and shown in stick configuration. Secondary structure of the cap subdomain is labeled and shown in red. (B) Ribbon representation of the dimeric C2221 crystal form of the Pf2001 esterase. Ser149 is labeled and shown in stick configuration. Secondary structure of the cap subdomain is labeled and shown in red. (C) Ribbon representation of the three α helices of the cap domain in the monomer of Pf2001. Hydrophobic patch residues are shown in red stick representation and labeled. The canonical esterase domain is shown in surface representation (blue surface). (D) Ribbon representation of the two α helices and the N-terminal β strand of the cap domain in the dimer of Pf2001. Hydrophobic residues are shown in blue stick representation and labeled. The canonical esterase domain is shown in surface representation (red surface). Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Structural Details of the Dimer Interface of the Pf2001 Esterase (A) Top: secondary structure representation of Pf2001 dimer in the C2221 and P212121 crystal forms. Secondary structure elements participating in the dimer interface are colored blue and red. Bottom: close-up view of some interface residues shown in stick representation (for specific contact details, see Table S1). (B) Top: secondary structure representation of Pf2001 dimer in the P21 crystal form. Secondary structure elements participating in the dimer interface are colored blue and red. Bottom: close-up view of some interface residues shown in stick representation (for specific contact details, see Table S2). (C) Structural comparison of the two Pf2001 dimers after superposition of one of their subunits (light red subunit). Pivotal residue Trp194 from the two overlapping structures is labeled and shown in stick representation. The arrow indicates the rigid body movement to align the structures. Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Formation of the Substrate Channel in the Pf2001 Dimer (A) Transparent surface representation of the two molecules of the dimer (blue and yellow) forming the active-site substrate channel in the P21 crystal form. Active-site methyl thiohexanoate inhibitor is shown in stick configuration. Red arrow indicates the active-site substrate channel. (B) Transparent surface representation of the two molecules of the dimer (blue and yellow) forming the active-site substrate channel in the P212121 crystal form. Active-site PEG400 molecule is shown in stick configuration. Red arrow indicates the active-site substrate channel. (C) Stereo close-up view of the active-site channel in complex with methyl thiohexanoate. Active-site residues are labeled and shown in stick representation. Methyl thiohexanoate inhibitor is labeled as Mth. The opposite molecule in the dimer forming the active site is represented as a transparent blue surface. (D) Stereo close-up view of the active-site channel in complex with a PEG400 molecule. Active-site residues are labeled and shown in stick representation. The opposite molecule in the dimer forming the active site is represented as a transparent blue surface. (E) Close-up view of the ribbon representation of the "open" (left) and "closed" (right) channels of the two active sites in the P212121 crystal form of the Pf2001 dimer. Relevant residues and PEG400 molecule are labeled and shown in stick configuration. Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Two Different Active-Site Conformations in the Dimer of the Pf2001 Esterase Ribbon representation of the dimer of the P212121 crystal form with a PEG400 molecule in one of the two active sites. PEG400 molecule is labeled as P400. Right top: stereo close-up view of one active site in complex with PEG400. Active-site residues are labeled and shown in stick configuration. The opposite molecule in the dimer forming the active site is represented as a transparent blue surface. Right bottom: stereo close-up view of the empty active site of the dimer. Active-site residues are labeled and shown in stick configuration. The opposite molecule in the dimer forming the active site is represented as a transparent red surface. Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Schematic Representation of the Dimerization Process of the Pf2001 Esterase Hypothetical transition between monomer (left) and two different types of dimers (center and right) observed in four different crystal forms of the Pf2001 esterase. Dark gray figure represents the canonical esterase domain. Light figures represent the secondary structure elements in the cap subdomain. AS and Nt labels depict the active-site region and the N terminus, respectively. Black arrow indicates the active-site channel predicted from the crystal structures of the P212121 and P21 dimers. Structure 2018 26, 199-208.e3DOI: (10.1016/j.str.2017.12.004) Copyright © 2017 Elsevier Ltd Terms and Conditions