Conserved conformational dynamics determine enzyme activity

Abstract

Homologous enzymes often exhibit different catalytic rates despite a fully conserved active site. The canonical view is that an enzyme sequence defines its structure and function and, more recently, that intrinsic protein dynamics at different time scales enable and/or promote catalytic activity. Here, we show that, using the protein tyrosine phosphatase PTP1B, residues surrounding the PTP1B active site promote dynamically coordinated chemistry necessary for PTP1B function. However, residues distant to the active site also undergo distinct intermediate time scale dynamics and these dynamics are correlated with its catalytic activity and thus allow for different catalytic rates in this enzyme family. We identify these previously undetected motions using coevolutionary coupling analysis and nuclear magnetic resonance spectroscopy. Our findings strongly indicate that conserved dynamics drives the enzymatic activity of the PTP family. Characterization of these conserved dynamics allows for the identification of novel regulatory elements (therapeutic binding pockets) that can be leveraged for the control of enzymes.

Fig. 1.. Coevolutionary coupling analysis identifies a conserved domain that influences PTP1B activity.

(A) Overlay of the open [Protein Data Bank (PDB): 5K9V; WPD loop only) and closed (PDB: 5K9W; gray cartoon) states of PTP1B. Highlighted functional elements include the PTP and Q loops (red), the WPD loops (closed, light orange; open, orange), the E loop (pink), helix α3 (lavender) and helix α4 (light blue), and the allosteric pocket (green circle). (B) Evolutionary partitioning (quality score versus Q divisions) identifying groups of coevolving residues (ED) in PTP1B using multiple sequence aålignments (MSA). (C) PTP1B EDs identified for Q = 4: ED1, N terminus (beige); ED2, core PTP domain (gray); ED3, β strands 7 to 10 (lavender); ED4, allosteric pocket (green). (D) PTP1B EDs identified for Q = 6: ED1 and ED4 same as in (C); ED2a, key catalytic loops (light gray); ED2b, support helices α4/α5 and part of helix α6 (dark gray); ED3a, β strands β9/β10 (with parts of β4 and β8) that are now grouped with helix α3 and loop L14 (blue); ED3b, the remainder of β strands β8 and β7 (lavender). Orange circle highlights the position of F225 on helix α4, which is part of ED3b. (E) ED3a hydrophobic pocket centered on F225. (F) Catalytic activity of ED3a variants (n = 3 to 4). (G) Kinetic parameters for measurements in (F).

Fig. 2.. Modulation of PTP1B activity by ED3a.

(A) Catalytic activity for PTP1B∆7 variants (n = 3 to 4). (B) Overlay of the 2D [¹H,¹⁵N] TROSY spectrum of PTP1B (black) and PTP1B_F225Y (blue). (C) CSPs of PTP1B versus PTP1B_F225Y (top) or PTP1B_{F225Y-R199N-L195R} (bottom). Red bars indicate peaks that broadened beyond detection; PTP1B secondary structural elements are shown, and the location of mutations is indicated by a diamond. (D) Loop L14 and L204 and its relationship to ED3a (residues F225, L195, and R199) and the active site PTP loop (C215). Structural elements are colored as in Fig. 1A. ppm, parts per million.

Fig. 3.. Group 2 dynamics are related to enzyme catalysis.

(A) Catalytic activity for PTP1B L14 variants (n = 3 to 4). (B) Overlay of ¹³C ILV 2D [¹H,¹³C] HSQC spectrum of PTP1B (black) and PTP1B_L204A (green). Moving peaks are annotated and indicated with arrows. (C) Representative ¹³C-CPMG dispersion profiles for V155 [fast exchange in free PTP1B_L204A (black) and intermediate exchange in TCS401-saturated PTP1B_L204A (yellow)] and L227 [free PTP1B_L204A (black) and intermediate exchange in TCS401-saturated PTP1B_L204A (yellow)]. (D) Left: ct-CPMG relaxation dispersion grouping for PTP1B_L204A (PDB 7MNC), group 1 (k_ex = 3000 ± 40 s⁻¹; dark blue) and group 2 (k_ex = 5000 ± 210 s⁻¹; orange). Right: ct-CPMG dispersion relaxation grouping for PTP1B_L204A saturated with TCS401 (PDB 7MND), group 1 (k_ex = 3130 ± 280 s⁻¹; blue), group 2 (k_ex = 1340 ± 120 s⁻¹; yellow), and group 3 (k_ex = 680 ± 40 s⁻¹; raspberry). (E) ct-CPMG dispersion relaxation group 2 in TCS401-saturated PTP1B (left) (24) and TCS401-saturated PTP1B_L204A (right). The exchange frequency (k_ex) and k_AB, along with the measured k_cat, for TCS401-saturated PTP1B and PTP1B_L204A are shown. (F) Group 2 residues (yellow sticks) cradle helix α4 (residues that anchor the PTP loop are shown in dark yellow). The PTP loop, including the catalytic C215, is shown in red.

Fig. 4.. Group 2 residues are highly conserved.

(A) Overlay of PTP1B (white), TCPTP (light gray), STEP (dark gray), and HePTP (black). Residues surrounding helix α4 are shown as sticks and colored by conservation within the PTP family. Left: Front view looking down helix 4 from the active site (yellow star; catalytic Cys shown in orange sticks). Pink arrow indicates the view of the hydrophobic platform (curved magenta line) shown in (B). Right: Same overlay rotated by 180°. (B) PTP1B is shown as a surface and colored according to PTP family sequence conservation [most conserved (magenta) and least conserved (teal)]. Left: Same orientation as in (A), left. Right: Same orientation as in (A), right. Middle: View of helix α4 looking down illustrating the conserved hydrophobic platform and the increased variability at the α3-α4 junction. (C) Residues (shown as sticks) that comprise the conserved hydrophobic platform that support helix α4 are colored according to conservation and labeled (underline indicates a group 2 residue). (D) Residues (shown as sticks) illustrating the increased variability at the C-terminal portion of helix α4 adjacent to the N terminus of helix α3. Residues are colored and labeled as in (C).