Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2

Abstract

Lysyl oxidase−2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)−dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post−translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+−bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ−containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2−hydrazinopyridine (2HP). Comparative UV−vis and resonance Raman spectroscopic studies of the 2HP−inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP−modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent−exposed.

A 3D–Predicted Structure of the Amine Oxidase Domain of Lysyl Oxidase–Like 2

“…The C2 oxygen atom of TPQ–2HP (Tyr689) was removed to generate DPQ689 and was covalently modified with 2HP (DPQ–2HP) and then the ϵ–amino group of Lys653 was manually crosslinked to the C2 of DPQ–2HP to generate LTQ–2HP via a usage of the patch function of CHARMM [ 29 ]. The parameters for the patch residue were first predicted by CHARMM General Force Field (CGenFF), based on the X–ray crystal structure of the corresponding Cu 2+ –bound LTQ–2HP model compound ( Figure S1B ) [ 25 , 30 ]. While the structure of LTQ–2HP was successfully engineered in LOXL2, the –(CH 2 ) 4 –NH– side chain of Lys653 was forced to adopt an unfavorable rotamer in higher energy ( Figure 6 A).…”

“…The O4 (oxoanion) of LTQ–2HP is 2.5Å from Cu 2+ which is 0.2–0.3Å longer than other ligands to Cu 2+ at the Jahn–Teller axis. The X–ray crystal structure of Cu 2+ –ligated LTQ–2HP model compound revealed that Cu 2+ is in a distorted square pyramidal geometry with an Addison parameter (τ) of 0.004, where two bridging chloride ligands likely correspond to His628 and His630 in 2HP–inhibited LOXL2 ( Figure S1B ) [ 25 , 30 ]. At each frame during MD–simulation, the τ values in the average of 0.195 ± 0.003 and the maximum of 0.387 were calculated ( Figure 8 Β), supporting the hypothesis that the Cu 2+ coordination geometry is distorted square pyramidal.…”

“… The LTQ cofactor resides within 2.9Å of the active site Cu 2+ in the catalytically–competent LOXL2 [ 25 ]. ( A ) 2HP covalently modifies the C5 carbonyl group of LTQ to form LTQ–2HP adduct.…”

“…These results support our hypothesis that a substantial conformational rearrangement is not required for LTQ biogenesis. Further, the UV–vis and resonance Raman spectroscopic features of 2HP–inhibited LOXL2 in comparison to the corresponding model compounds strongly suggested that 2HP covalently modifies LTQ (LTQ–2HP), and that LTQ–2HP is ligated to the active site Cu 2+ ( Figure 2 A) [ 25 ], analogous to the 2HP–modified topaquinone (TPQ) cofactor (TPQ–2HP) detected in a crystal structure of Y369F–ECAO (a CAO from Escherichia coli , E. coli ) ( Figure S1A ) [ 26 ]. Interestingly, LTQ–2HP contains the structural motif similar to 4–(2–pyridylazo)resorcinol (PAR), a known chelator.…”

“…Interestingly, LTQ–2HP contains the structural motif similar to 4–(2–pyridylazo)resorcinol (PAR), a known chelator. We found that the active site Cu 2+ in the mature LOXL2 can be fully titrated by PAR ( Figure 2 B) [ 25 ]. Based on these results, we believe that the LTQ cofactor resides within 2.9Å from the active site Cu 2+ in the mature LOXL2 and both LTQ and Cu 2+ are solvent–exposed as in the case of the precursor Tyr689 and Zn 2+ in the precursor.…”