Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

Abstract

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination.

Keywords: NAD(P)H-dependent reductase; Rossmann fold; X-ray crystallography; analytical ultracentrifugation; enzyme kinetics; nicotinamide adenine dinucleotide (NADH); reductase; site-directed mutagenesis; substrate specificity.

Figure 1.

The reactions and enzymes of proline biosynthesis. G5K, glutamate-5-kinase; γ-GPR, γ-glutamate-phosphate reductase; OAT, ornithine-γ-aminotransferase.

Figure 2.

Structures of the PYCR1 protomer and dimer. A, structure of the protomer of the ternary complex with NADPH and the proline/P5C analog THFA. The N-terminal NAD(P)H-binding domain is colored according to secondary structure, with β-strands in pink and α-helices in blue. The C-terminal oligomerization domain is colored gray. NADPH appears in gold sticks. THFA is shown as cyan sticks. β-Strands are labeled 1–8; α-helices are labeled A–M. Helix-disrupting Pro-178 is shown. B, structure of the dimer. The α-helices of the C-terminal domain are labeled H–M for the gray protomer and H′–M′ for the purple protomer. NADPH and THFA are colored gold and cyan, respectively. The arrow represents the 2-fold axis of the dimer.

Figure 3.

Oligomerization of PYCR1 in solution. A, apparent sedimentation coefficient distribution determined at 6 mg/ml (solid line) and 0.8 mg/ml (dashed line). B, molecular mass distribution determined at 6 mg/ml (solid line) and 0.8 mg/ml (dashed line).

Figure 4.

The PYCR1 pentamer-of-dimers decamer. A, two orthogonal views of the decamer, with each chain differently colored. B, two orthogonal views of the decamer, with the N-terminal domains colored blue and the C-terminal domains colored gray. Note that the C-terminal domains mediate all protein-protein interactions in the decamer.

Figure 5.

Electron density and interactions for NADPH bound to PYCR1. The cage represents a simulated annealing F_o − F_c map contoured at 3σ. Selected α-helices and β-strands are labeled in accordance with Fig. 2A. Helix K (purple) is from the opposite protomer of the dimer. The conserved water molecule of the Rossmann dinucleotide-binding fold is colored green (21).

Figure 6.

The proline-binding site. A, electron density and interactions for proline (cyan) bound to PYCR1. The cage represents a simulated annealing F_o − F_c map contoured at 3σ. The two protomers of the dimer are colored purple and gray. Note that proline binds in the dimer interface. Selected α-helices are labeled in accordance with Fig. 2A. B, space-filling representation of the proline-binding site highlighting nonpolar residues that contact the methylene groups of proline.

Figure 7.

Structure of the ternary complex of PYCR1 with NADPH and THFA. The cage represents a simulated annealing F_o − F_c map contoured at 3σ. NADPH and THFA are colored gold and cyan, respectively. The two protomers of the dimer are colored purple and gray. Two orthogonal views are shown. Selected α-helices are labeled in accordance with Fig. 2A.