Why Bestatin Prefers Human Carnosinase 2 (CN2) to Human Carnosinase 1 (CN1)

Abstract

Human carnosinases (CNs) are Xaa-His metal-ion-activated aminopeptidases that break down bioactive carnosine and other histidine-containing dipeptides. Carnosine is a bioactive peptide found in meat and prevalently used as a supplement and in functional food formulation. Nonetheless, carnosine is digested by CNs rapidly after ingestion. CNs have two isoforms (carnosinase 1 (CN1) and carnosinase 2 (CN2)), where CN1 is the main player in carnosine digestion. CNs contain a catalytic metal ion pair (Zn²⁺ for CN1 and Mn²⁺ for CN2) and two subpockets (S1 and S1' pockets) to accommodate a substrate. Bestatin (BES) has been reported to be active for CN2; however, its inhibition ability for CN1 has remained under debate, because the underlying mechanism remains unclear. This information is important for designing novel CN1-selective inhibitors for proliferating carnosine after ingestion. Thus, molecular dynamics (MD) simulations were performed to explore the binding mechanism of BES to both CN1 and CN2. The binding of BES-CN1 and BES-CN2 was studied in comparison. The results indicated that BES could bind both CNs with different degrees of binding affinity. BES prefers CN2 because: (1) its aryl terminus is trapped by Y197 in an S1 pocket; (ii) the BES polar backbone is firmly bound by catalytic Mn²⁺ ions; and (iii) the S1' pocket can shrink to accommodate the isopropyl end of BES. In contrast, the high mobility of the aryl end and the complete loss of metal-BES interactions in CN1 cause a loose BES binding. Seemingly, polar termini were required for a good CN1 inhibitor.

Figure 1

(A) Structure of human orthologues of CN1 and CN2 homodimer with two catalytic metal ions (Zn²⁺ in CN1 and Mn²⁺ in CN2) and bestatin (orange) where the catalytic and dimerization domains are also labeled. The latching loops (L2) are displayed in green. The active site is shown in (B). Superimposition of BES (dark pink in CN1 and light pink in CN2) and pocket-lining residues of CN1 (dark orange/cyan) and CN2 (light orange/cyan) are displayed. The S1 and S1′ pockets are labeled in brown and cyan, respectively. The chemical structure of BES is shown on the top right with the active oxygen (O3) labeled.

Figure 2

C-α RMSDs (A) and RMSFs (B) of all systems. (C) Superimposition of CN1 and CN2 from human orthologues with the latching loop (L2 in green) and catalytic metal ions. The highly flexible loops are displayed in red, blue, magenta, and green.

Figure 3

(A) Principal Component Analysis (PCA) of all of the CNs. The motions were obtained from the first principal component (PC1) as a function of time, which are represented in RGB format. Only dominant motions are displayed for clear visualization. (B) Distances between the C-α atoms of D78-P333 in CN1, and D71-S362 in CN2. The locations of these two residues are shown in (C) where L1 and L2 are labeled in red and green.

Figure 4

Number of hydrogen bonds of CN1-BES (A) and CN2-BES (B) as a function of time. The locations of key residues are displayed on the right.

Figure 5

(A) Conformational changes of BES and metal ions as a function of time. The red dashed line indicates the distance between a metal pair, where the distance with the standard deviation is also displayed in red. (B) Alignment of the BES structure in the active site (CN1 on the left and CN2 on the right). Orange and violet surfaces indicate the S1 and S1′ pockets where the aromatic moiety points toward the S1 pocket and the isopropyl end is inserted into the S1′ pocket.

Figure 6

(A) Structures of metal ion pairs in complex with coordinating residues and water molecules in all systems. The black solid line indicates the interaction distance between each metal ion and its adjacent residues (<2.0 Å for Zn²⁺ and <2.4 Å for Mn²⁺). These interaction distances were identified elsewhere.⁻ The black dashed line shows the metal–residue distance which is further away from metal ions (>2.0 Å for Zn²⁺ and >2.4 Å for Mn²⁺). (B) Distances of metal-O3 and metal–OH in all systems. The positions of O3, OH, and metal ions are also shown on the top.