Enzyme inhibition by allosteric capture of an inactive conformation

Abstract

All members of the human herpesvirus protease (HHV Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-Å-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions.

Fig. 1. Domain diagram of KSHV Pr

(a) Linear domain diagram of KSHV Pr displaying the positions of the “hot spot” Trp109 (red), catalytic residues (cyan) and the conformationally dynamic C-terminus (gray). C-terminal truncations are indicated by yellow or blue asterisks. (b) The dimer interface of a KSHV Pr monomer (2PBK). The partner monomer is omitted for clarity. The active site (cyan), the inhibitor-binding “hot spot” Trp109 (red), and truncation sites (yellow and blue balls) are indicated as in Figure 1a. See also Movie S1. (c) The chemical structure of DD2, an optimized analog of a first generation lead inhibitor of KSHV Pr.

Fig. 2. KSHV Pr-DD2 titration

(a) The distribution of isoleucine δ1-methyl groups on a full length KSHV Pr monomer structure, based upon 2PBK. The view is a 90 degree rotation about the horizontal axis, relative to that in Figure 1b. Isoleucines separate into five zones with respect to distance from Trp109 as indicated by colored balls. The ¹³C-¹H HSQC titration spectra of the KSHV Pr M197D (b) and Δ196 (c) constructs with DD2, focusing on the isoleucine δ1-methyl region. The spectral overlays display apo (black) and > 5 molar equivalents DD2 (red). Ile44 (solid blue box), Ile105 (dotted blue box), and Ile71 (solid black box) are used as the binding probes. (d) The binding curves (M197D, solid black; Δ196, dashed red) represent the average apparent K_d values calculated for the three Ile probes, and are summarized as a bar chart (e). HSQC titration spectra and binding curves for the M197D-I201V construct are displayed in Figure S4.

Fig. 3. Structural comparison of the “apo” and DD2-inhibited KSHV Pr monomers

(a) The dimer structure of peptide-phosphonate inhibited KSHV Pr (2PBK). The catalytic residues (cyan) are located ~ 15 – 20 Å from the dimer interface. The interfacial helix 5 and the following helix 6 (monomer A, red; monomer B, green) are displayed. Helix 1 of monomer A (blue) and monomer B (orange) also form a portion of the dimer interface and are aligned in an anti-parallel orientation with respect to each other. (b) The structure of the KSHV Pr Δ196-DD2 complex (3NJQ) crystallizes as an asymmetric dimer, with dimerization occurring on the opposite face with respect to 2PBK. DD2 molecules bind to the hydrophobic surface normally occupied by helix 5. Monomer A of the complex contains one DD2 molecule (pose 1, green carbons), while monomer B contains two DD2 molecules (pose 2, magenta carbons; pose 3, cyan carbons). The truncated C-terminal residues of the Δ196 constructs (red, monomer A; green, monomer B) are also indicated. Helix 1 of monomer A (blue) and monomer B (orange) are oriented end-on-end with respect to each other. Below each structure are cartoon representations of the monomeric units, with the wedges representing the active site, and stars the DD2 molecules.

Fig. 4. Comparison of the apo and DD2-bound KSHV Pr crystal structures

(a) The dimer interface of KSHV Pr (2PBK) consists of two helices from each monomeric unit (helix 5, tan, monomer A; light blue, monomer B), which stabilize the active site (cyan) via the C-terminal helix 6 and occlude the Trp109 (red). (b) The Met197 and Ile201 sidechains from helix 5 of monomer B (orange) form hydrophobic interactions with Trp109 of monomer A. Both Δ196-DD2 monomer A (c) and monomer B (Fig. S5) exhibit independent DD2 binding pockets in which the Trp109 sidechain indole ring (red) adopts an “open” form (d). See also Movie S2.

Fig. 5. The DD2 binding pocket

(a) The hydrophobic DD2 binding pocket is composed of aliphatic residues from the β2-β3 loop (yellow), helix 1 and the α1-α2 loop (blue), helix 2 (red), the β6-β7 loop (orange), and the C-terminus (magenta). DD2 (green carbons) is shown as a space-filling model. (b) Stereoview of DD2 (green) within the Δ196 binding pocket of monomer A, in relation to the “hot spot” Trp109 (red) and the Ile44 and Ile105 reporter groups (yellow). Also displayed are buried aliphatic residues of helix 1 (blue), helix 2 (red), and the C-terminus (magenta) that compose the binding pocket. The mesh represents the 2F_o-F_c 1σ electron density map. (c) Figure 5b with the protein backbone ribbons displayed. Comparable views of monomer B are displayed in Figure S5.

Fig. 6. The conformations of KSHV Pr-bound DD2 in situ

(a) DD2-A (green) and DD2-B (magenta) within the Δ196-DD2 complex are overlaid with Helix 5 of the KSHV Pr partner monomer B (cyan). Orange balls represent the non-branched methyl groups of Met197 and Ile201. The DD2 “sidechains” match the relative positions observed for those of the native Met197 and Ile201, and are inserted into the hydrophobic pocket vacated by the Trp109 indole ring. The protein monomer structures of the Δ196-DD2 complexes and the apo KSHV Pr dimer are omitted for clarity. (b) An overlay of Δ196-DD2 monomers with their constituent DD2 molecules. Monomer A (gray) contains DD2-A (green), while monomer B (dark blue) contains DD2-B (magenta) and DD2-C (cyan). DD2-C is an artifact of crystal packing and is situated outside the DD2 binding pocket. Major conformational differences in the DD2 binding pocket are only observed for the C-terminal residues (Ser191 – Leu196). (c) An overlay of Δ196-DD2 monomers with their respective DD2 molecules displaying the sidechains of the residues constituting the DD2 binding pocket. The benzyl “sidechain” of DD2-A and DD2-B are in close proximity to Phe76, while the cyclohexyl ring interacts with Ile105, Leu106, and Leu110.

Fig. 7. Structural perturbation of the KSHV Pr active site upon DD2 binding

(a-b) The active site of the 2PBK represents an “apo” state of KSHV Pr. The catalytic triad (H46, H134, and S114, cyan) and the conserved oxyanion hole-stabilizing arginine residues (R142 and R143, red) are displayed as sticks. Also highlighted are the positions of β-strand 1 and α-helix 0 (yellow), the β1/α0 loop (dark green), and the β6/β7 loop (orange). Residues 197–230 are omitted for clarity. (c-d) The conformation of the “apo” state active site residues displays clear differences relative to the Δ196-DD2 complex (3NJQ). The Arg142 and Arg143 sidechains (red) adopt a “closed” conformation in the apo state, but an “open” conformation while in complex with DD2. In the DD2-bound state, the β1/α0 loop (dark green) occludes the catalytic triad (cyan) and disrupts the substrate binding pocket. See also Movie S3.

Fig. 8. CD spectra of DD2 titrations with KSHV Pr and HCMV Pr

The circular dichroism spectra of ~ 3 μM (a) KSHV Pr and (b) HCMV Pr in the presence of 0 μM (black), 30 μM (red), and 80 μM (blue) DD2. Estimated fractional helicity (f_H) values derived from the mean residue ellipticity of the 222 nm band are listed in the insets, and indicate loss of helical content with increasing molar equivalents of DD2. Loss of helicity is a strong indication of HHV protease dimer disruption.

Fig. 9. HCMV Pr-DD2 titration data

(a) The three isoleucine δ1-methyl groups in human CMV Pr are localized at the dimer interface, and color-coded with respect to distance to Tyr128, as indicated. Helix 5 (tan), the active site (cyan), and Tyr128 (red) are also displayed. Tyr128 is homologous to Trp109 of KSHV Pr. The ¹³C-¹H HSQC spectra of selective [¹³C-¹H methyl] isoleucine labeled CMV Pr L222D obligate monomer (b), and Δ221 truncation (c) in the presence of 0 (black) and 16 molar equivalents DD2 (red) indicates DD2 binds at the dimer interface. Both Ile61 and Ile96 are putatively assigned; Ile231 was assigned by the loss of the resonance in the Δ221 truncation.

Fig. 10. Structural homology of the HHV protease “hot spots”

(a) Representative X-ray crystallographic structures of the three structurally homologous HHV protease subfamilies (HSV-2 Pr, 1AT3; HCMV Pr, 1CMV; KSHV Pr, 2PBK), with the active site (cyan) and interfacial helix 5 and following helix 6 (tan) as indicated. “Hot spot” aromatic residues (red) are located at the center of the hydrophobic dimer interface and are potential target sites for small-molecule inhibitors that disrupt protein-protein interactions. α-subfamily HHV proteases: HSV-1 = herpes simplex virus-1/; HSV-2 = herpes simplex virus-2; VZV = Varcella Zoster virus. β-subfamily HHV proteases: HCMV = human cytomegalovirus; HHV-6 = human herpesvirus-6; HHV-7 = human herpesvirus-7. γ-subfamily HHV proteases: KSHV = Kaposi’s sarcoma-associated herpesvirus; EBV = Epstein-Barr virus. (b) Backbone overlay of KSHV Pr (2PBK, gray) and HSV-2 Pr (1AT3, cyan), focusing on the dimer interface “hot spot” region. The sidechains of Trp109 (KSHV Pr, red) and Tyr124 (HSV-2 Pr, yellow) are displayed in space-filling mode. Helices 5 and 6 are omitted for clarity. (c) Backbone overlay of KSHV Pr (2PBK, gray) and HCMV Pr (1CMV, cyan), focusing on the dimer interface “hot spot” region. The sidechains of Trp109 (KSHV Pr, red) and Tyr128 (HCMV Pr, yellow) are displayed in space-filling mode. Helices 5 and 6 are omitted for clarity.