Insights on the conformation and appropriate drug-target sites on retinal IMPDH1 using the 604-aa isoform lacking the C-terminal extension

Abstract

Background and purpose: Retinitis pigmentosa (RP) accounts for 2 percent of global cases of blindness. The RP10 form of the disease results from mutations in isoform 1 of inosine 5'-monophosphate dehydrogenase (IMPDH1), the rate-limiting enzyme in the de novo purine nucleotide synthesis pathway. Retinal photoreceptors contain specific isoforms of IMPDH1 characterized by terminal extensions. Considering previously reported significantly varied kinetics among retinal isoforms, the current research aimed to investigate possible structural explanations and suitable functional sites for the pharmaceutical targeting of IMPDH1 in RP.

Experimental approach: A recombinant 604-aa IMPDH1 isoform lacking the carboxyl-terminal peptide was produced and underwent proteolytic digestion with α-chymotrypsin. Dimer models of wild type and engineered 604-aa isoform were subjected to molecular dynamics simulation.

Findings/results: The IMPDH1 retinal isoform lacking C-terminal peptide was shown to tend to have more rapid proteolysis (~16% digestion in the first two minutes). Our computational data predicted the potential of the amino-terminal peptide to induce spontaneous inhibition of IMPDH1 by forming a novel helix in a GTP binding site. On the other hand, the C-terminal peptide might block the probable inhibitory role of the N-terminal extension.

Conclusion and implications: According to the findings, augmenting IMPDH1 activity by suppressing its filamentation is suggested as a suitable strategy to compensate for its disrupted activity in RP. This needs specific small molecule inhibitors to target the filament assembly interface of the enzyme.

Keywords: Inosine monophosphate dehydrogenase; Molecular dynamics simulation; Proteolytic digestion; Retinal isoforms; Retinitis pigmentosa.

Fig. 1

Proteolysis of (A) wild type and (B) ΔC engineered 604-aa IMPDH1 isoforms. The proteolytic digestion was followed for 0, 2, 4, 6, 8, 10 and 15 min. IMPDH1; Inosine 5'-monophosphate dehydrogenase isoform 1; ΔC, the 604-aa isoform lacking the C-terminal peptide.

Fig. 2

RMSD and RMSF plots for wild type and engineered retinal IMPDH1 modelled as dimers. (A) RMSD plot of the wild type (WT-604) and engineered protein (Eng-604); (B) RMSF plot for individual protomers of wild type (WT-1 and WT-2) and engineered (Eng-1 and Eng-2) dimers. RMSD, Root mean squared deviation; RMSF, root mean squared fluctuation; IMPDH1, inosine 5'-monophosphate dehydrogenase isoform 1.

Fig. 3

Interactions between catalytic flap (magenta) and C-terminal peptides (blue) of distinct wild type 604-aa isoforms. (A) Overall view of interactions between dimers; (B) detailed view of hydrogen bonds between C-terminal peptide and catalytic flap of distinct monomers. Hydrogen bonds formed between T577, C576, and S565 of the C-terminal peptide and G472, S473, and D475 of the catalytic flap, respectively. To simplify the visualization of interactions, the N-terminal peptide was omitted.

Fig. 4

(A) Secondary structural illustration for IMPDH1 604-aa isoform in its wild type (left) and ΔC engineered (right) forms. A new helix (marked with an arrow) was formed in the wild type, but not in the absence of a C-terminal peptide in the ΔC recombinant; (B) formation of a short helix in the GTP2 binding site. Magenta: N-terminal peptide; green: Asp283; orange: GTP. IMPDH1; Inosine 5'-monophosphate dehydrogenase isoform 1; ΔC, the 604-aa isoform lacking the C-terminal peptide; GTP, guanosine triphosphate; CBS, cystathione β-synthase.

Fig. 5

(A) Interactions of N- and C-terminal peptides within the same monomer of wild type 604-aa isoform (magenta: N-terminal peptide and blue: C-terminal peptide); (B) catalytic flaps (magenta and cyan) of distinct monomers of the engineered protein interact in the absence of the C-terminal peptide.