Ab Initio Modelling the Structure of Proton-Sensing G-Protein Coupled Receptor GPR151

: Protein is the proteios building block of life. Evolutionarily, its sequence is not as conserved as its structure, making it more reasonable for protein structure, instead of protein sequence, to be the descriptor of protein function. Yet, in the National Center for Biotechnology Information (NCBI) database, the number of experimentally identiﬁed protein sequences is in great excess of that of experimentally determined protein structures inside the almost-half-a-century old Protein Data Bank (PDB). For instance, GPR151 is an proton-sensing G-protein coupled receptor (GPCR) originally identiﬁed as homologous to galanin receptors. As of March 19, 2020, GPR151’s structure has not been experimentally determined and deposited in PDB yet. Thus, an ab initio modelling approach was employed here to build a three-dimensional structure of GPR151. Overall, the ab initio GPR151 model presented herein constitutes the ﬁrst structural hypothesis of GPR151 to be experimentally tested in future with previously published, currently ongoing and future GPR151 studies. structural modelling processes.


Introduction
It has been almost half a century since the launch of Protein Data Bank (PDB) in 1971 [1]. Biophysical tools such as X-ray crystallography, NMR spectroscopy and Cryo-electron microscopy have contributed enormously to the continued development of PDB [2], with which a variety of computational tools have been developed for biomolecular structural modelling, classification and feature extraction [3][4][5][6][7][8][9][10][11][12] and functional prediction. For instance, ab initio protein structural modelling [13,14] is an energy function-guided method to predict protein structure in the absence of experimentally solved structure of a similar/homologous protein. Also termed as de-novo modelling, physics-based modelling or free modelling, the ab initio approach is often preferred for structure prediction when there is no or very low amount of similarity for the query protein sequence.

Motivation
Protein is the proteios building block of life. Evolutionarily, its sequence is not as conserved as its structure, making it more reasonable for protein structure, instead of protein sequence, to be the descriptor of protein function. Yet, in the NCBI database, the number of experimentally identified protein sequences is in great excess of that of experimentally determined protein structures inside the almost-half-a-century old PDB [1]. For instance, GPR151 is an proton-sensing G-protein coupled receptor (GPCR) originally identified as homologous to galanin receptors [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30].
As of March 19, 2020, GPR151's structure has not been experimentally determined and deposited in PDB yet.
Furthermore, with a structural search by the SwissModel server (https://swissmodel.expasy.org/interactive) [31], it turned out that there is no experimentally determined structure of GPR151-homologous protein in PDB [1], and that the highest similarity possible for GPR151 is only 17.33% for the chain A of Neurotensin receptor type 1 (PDB ID: 4XES) as of March 19, 2020. As a result, homology structural modelling is not an option feasible here for GPR151. Therefore, this article employs an ab initio modelling approach to build a three-dimensional structure of GPR151.

Materials and Methods
With a set of online resources (https://www.ncbi.nlm.nih.gov/protein/EAW61839.1 and https://www.uniprot.org/uniprot/Q8TDV0), the amino acid sequence of GPR151 was retrieved and listed below, The ab initio construction of GPR151's structure started from its sequence consisting of 419 amino acid residues as above, and employed the Quark ab initio modelling server (https://zhanglab.ccmb.med.umich.edu/QUARK/) [13,14], which was ranked as the No. 1 server in Free-modelling (FM) in CASP9 and CASP10 experiments, making it suitable for proteins that do not have homologous templates (like GPR151) in the PDB database [1].
Given that Quark does not accept job submission with protein sequence longer than 200 amino acid residues, the GPR151 sequence is thus manually separated into three fragments for three ab initio structural modelling processes.

SDPAKQVSIHNYTIWSVLVAIWTVA S L L P L P E W F F S T I R H H E G V E M C L V D V P A V A E E FMSMFGKLYPLLAFGLPLFFASFYFWRAYDQCKKRGTKTQNLRNQIRSKQVTVM L L S I A I I S A L L W L P E W V A W L W V W H L K A A G P A P P Q G F I A L S Q V L M F S I S S A N P L I F L VMSEEFREGLKGVWKWMITKKPPTVSESQET 3. MSEEFREGLKGVWKWMITKKPPTVSESQETPAGNSEGLPDKVPSPESPASIPEKEKPSSPSSGK GKTEKAEIPILPDVEQFWHERDTVPSVQDNDPIPWEHEDQETGEGVK
Specifically, the ab initio construction of GPR151's structure consists of seven steps as below, 1. The ab initio construction of GPR151's first fragment was performed with the QUARK server with an output PDB file (supplementary file model1.pdb). 2. The ab initio construction of GPR151's second fragment was performed with the QUARK server with an output PDB file (supplementary file model2.pdb). 3. The ab initio construction of GPR151's third fragment was performed with the QUARK server with an output PDB file (supplementary file model3.pdb). 4. An in-house python script (supplementary file k1.py) was used to dock model2.pdb (red fragment) to model1.pdb (red fragment), yielding an ab initio modelled structural fragment (from Met1 to Thr338) of GPR151 (supplementary file k1.pdb). 5. k1.pdb was subsequently subject to an energy minimization process on the ModRefiner server (https://zhanglab.ccmb.med.umich.edu/ModRefiner/) [32], yielding an ab initio modelled energy-minimized structural fragment (from Met1 to Thr338) of GPR151 (supplementary file k1refine.pdb). 6. An in-house python script (supplementary file k2.py) was used to dock k1refine.pdb (blue fragment) to model3.pdb (blue fragment), yielding an ab initio modelled full-length structure of GPR151 (supplementary file k2.pdb). 7. k2.pdb was subsequently subject to an energy minimization process on the ModRefiner server, yielding an ab initio modelled energy-minimized full-length structure of GPR151 (supplementary file k2refine.pdb)

Result and Conclusion
With the ab initio structural modelling steps described above, Figure 1 presents an overall view of the three-dimensional scaffold of GPR151. Figure 1. An ab initio structure of GPR151. In this figure, GPR151 is shown as rainbow-coloured cartoons, with its N-and C-termini coloured blue and red, respectively. This figure is prepared using PyMol [33] with supplementary file k2refine.pdb as an input.
The ab initio modelled atomic coordinates of GPR151 are included in the supplementary file k2refine.pdb, which does not include side chain hydrogen atomic coordinates for GPR151 residues. With Chimera [34], nonetheless, all side chain hydrogen atoms can be placed at their respective spatial locations where the GPR151 structural model corresponds to the global free energy minimum under a given set of conditions [35,36], such that no atomic detail is missing and that the ab initio modelled GPR151 structure is intact, i.e., experimentally uncharted territory (EUT)-less [3]. Overall, the ab initio GPR151 model here constitutes the first structural hypothesis of GPR151, which remains to be tested in future with all experimental GPR151 studies, previously published, currently ongoing and future ones, too.

Conflicts of Interest:
The author declares no conflict of interest.