Understanding the role of glucose regulated protein 170 (GRP170) as a nucleotide exchange factor through molecular simulations

Abstract

Glucose Regulated Protein 170 (GRP170), also called Oxygen Regulated Protein 150 (ORP150), is a major molecular chaperone resident in the endoplasmic reticulum (ER). It belongs to the heat shock protein (HSP70) super family and can be induced by conditions such as hypoxia, ischemia and interferences in calcium homeostasis. It was recently reported that GRP170 may act as a nucleotide exchange factor (NEF) for GRP78 or binding immunoglobulin protein (BiP), and the ER canonical HSP70. However, little is known about the mechanism underlying its NEF activity. In this study, two homology models of GRP170 were constructed based on the X-ray crystal structures of ADP and ATP bound HSP110, a cytosolic homolog of GRP170, in order to characterize the differences in the binding modes of both ligands. It was observed that the differences in the binding modes of ADP and ATP led to a conformation change in the substrate binding domain which could potentially influence the binding of its substrates such as BiP. Our findings help understand the effect of nucleotide binding on the function of this chaperone protein as a NEF as well as the structural differences between GRP170 and its family members.

Keywords: Docking; GRP170; Homology modeling; Nucleotide binding; Nucleotide exchange factor (NEF); ORP150.

Fig. 1.

Sequence alignment human GRP170 (UniProt ID Q9Y4L1) and yeast HSP110 (PDBID 3C7N, 2QXL). Identical residues are highlighted in red and structurally related residues are marked in red font.

Fig. 2.

A) Homology model of GRP170 in its active conformation (cyan) overlapped with the template structure (3C7N, magenta), TM-score 0.49; B) Homology model of GRP170 in its inactive conformation (grey) overlapped with the template structure (2QXL, green), TM-score 0.40; C) Crystal structures of ADP-bound (3C7N, magenta) and ATP-bound (2QXL, green) yeast HSP110 overlapped; D) The active (cyan) and inactive (grey) GRP170 homology models overlapped.

Fig. 3.

Ramachandran plots of the homology model of (A) active GRP170 and, (B) inactive GRP170 models

Fig. 4.

(A) ProSA-web z-scores of all proteins in PDB determined by X-ray crystallography (light blue) and NMR spectroscopy (dark blue) with respect to their length. The z-score of the active GRP170 model is shown as black dot and, (B) Energy plot of active GRP170 model. Energies are plotted as a function of the central residue in the window. A window size of 40 residues (dark green) and 10 residues (light green) was used.

Fig. 5.

(A) ProSA-web z-scores of all proteins in PDB determined by X-ray crystallography (light blue) and NMR spectroscopy (dark blue) with respect to their length. The z-score of the inactive GRP170 model is shown as black dot and, (B) Energy plot of inactive GRP170 model. Energies are plotted as a function of the central residue in the window. A window size of 40 residues (dark green) and 10 residues (light green) was used.

Fig. 6.

Most preferred docking pose of (A)ADP (backbone carbon atoms shown as green, nitrogen atoms as blue, oxygen atoms as red and phosphorous atoms as orange sticks) in the generated GRP170 homology model (cyan cartoon) and (B) ATP (backbone carbon atoms shown as magenta, nitrogen atoms as blue, oxygen atoms as red and phosphorous atoms as orange sticks) in the generated GRP170 homology model (grey cartoon). Backbone atoms of amino acid residues involved in interactions are shown as cyan/grey sticks with nitrogen atoms in blue and oxygen atoms in red.

Fig. 7.

A) Overlap of best scored docking pose of ADP (green) in the GRP170 active conformation homology model (cyan) and best scored docking pose of ATP (magenta) in the GRP170 inactive conformation homology model (grey); B) Overlap of ADP (green) and ATP (magenta) docking poses

Fig. 8.

The root-mean-square deviation (rmsd) of the protein backbone atoms of the ADP_GRP170^active and ATP_GRP170^inactive systems relative to the respective starting structures.

Fig. 9.

A) Overlap of active (cyan) and inactive (grey) homology models of GRP170 showing the change in the conformation of the amino acid residues; B) surface representation of active GRP170 homology model (cyan) and, C) surface representation of the inactive GRP170 homology model (grey). ADP (backbone carbon atoms shown as green, nitrogen atoms as blue, oxygen atoms as red and phosphorous atoms as orange sticks), ATP (backbone carbon atoms shown as magenta, nitrogen atoms as blue, oxygen atoms as red and phosphorous atoms as orange sticks) and backbone atoms of amino acid residues involved in interactions are shown as cyan/grey sticks with nitrogen atoms in blue and oxygen atoms in red.