Int J App Pharm, Vol 15, Issue 5, 2023, 287-293Original Article

STRUCTURAL PREDICTION OF HUMAN ZIP 2 AND ZIP4 BASED ON HOMOLOGY MODELLING AND MOLECULAR SIMULATION

GITA SYAHPUTRA1,3, NUNIK GUSTINI3, MELVA LOUISA2, MASTERIA YUNOVILSA PUTRA3, ADILAH FADILAH4*

1Doctoral Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia. 2Department of Pharmacology and Therapeutics, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia. 3Research Center for Vaccine and Drug, National Research and Innovation Agency, Cibinong-16911, Indonesia. 4Department of Medical Chemistry, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia
*Corresponding author: Adilah Fadilah; *Email: fadilah.msi@ui.ac.id

Received: 04 May 2023, Revised and Accepted: 04 Jul 2023

ABSTRACT

Objective: This study aimed to analyze the structural proteins of zinc transporters as the target for drug actions and their molecular interactions.

Methods: The present study is about the homology modelling and analysis of the zinc transporter function using the in silico molecular modelling method. Homology modelling predicts the 3D structure of a protein based on the sequence alignment with one or more template proteins of known structure. This study using in silico molecular modelling method, explains the 3D structure of human ZIP 2 and ZIP4 with Ramachandran Plot analysis, physical and chemical characteristics, transmembrane prediction with structural biology, and binding site prediction through molecular docking simulation.

Results: Based on the physicochemical properties of the 3D structure of the ZIP2 and ZIP4 proteins, each comprises 309 amino acids and 582 amino acids with pI values of 5.85 and 5.24. The amino acid composition analysis showed that both proteins contain many Leucine amino acids. The Ramachandran diagram concludes that both proteins are stable in the stereochemical conformation forming a secondary structure. The binding amino acids on ZIP2 include Glu281, His216, Ser284, and Arg46. The binding amino acids in ZIP4 include Gln148, Gln154, Thr155, His197, Ala138, and Lys157.

Conclusion: Establishment of the structure and function of human ZIP2 and ZIP4 as zinc transporters in cell membranes and prediction of ZIP2 and ZIP4 binding sites through molecular dcoking.

Keywords: 3D structure, Zinc transporter, Molecular modelling, Molecular docking, Structural biology

© 2023 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2023v15i5.48240. Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Zinc bioavailability is influenced by the concentration and amount of zinc that can successfully transmit through the small intestine membrane [1]. The gastrointestinal tract's zinc absorption factors include solubility, binding with other compounds, pH, and enzymatic reactions. The factors affect zinc absorption through the small intestine membrane e [2, 3]. Studies related to zinc bioavailability have not been studied, especially the effect of pancreatic secretion on zinc absorption and homeostasis.

The Zinc and Iron Regulated Transport Protein (ZIP) family of proteins is central to the transition homeostasis of metals. Their function is to increase the cytosolic concentrations of zinc and iron. There are 14 mammals ZIP family members, namely ZIP 1–ZIP 14 [4]. The ZIP family functions to facilitate cellular uptake or efflux from intracellular compartments. Studies show that ZIP2 is one of the key zinc transporters associated with zinc requirements during pregnancy. At the same time, ZIP4 and ZIP2 are transporter proteins that play a role in zinc absorption in the small intestine (duodenum) [5]. A mechanism that supports the description of ZIP transporters is the ZN2+/[HCO3-ion] [6, 7]. The transport mechanism of the ZIP family is not precisely known, so it is necessary to determine the model and function of ZIP2 and ZIP4 proteins.

Zinc transporters were also reported to be synthetic intracellular transducers that regulate cellular functions [8, 9], and various cytosol proteins can associate with zinc [10]. Conditions of physiological responses, such as developmental, immune, and neurological functions and the onset of several human diseases result from changes in zinc mineral concentration and intracellular distribution. The many reports related to zinc control in cells and its regulation make it important to know the biological function of the role of zinc transporters more deeply [11].

Determination of the protein model of zinc transporters ZIP2 and ZIP4 in the body's cellular function must be understood through the protein structural modelling. However, the protein database (PDB) used to find the protein structure is sometimes only partially complete. Therefore, a homology modelling method is needed to determine the comparative protein structure model from the query protein. The molecular docking method used in this study is determining the predicted binding sites of ZIP2 and ZIP4 while interacting with zinc carnosine as a third-generation zinc supplement [12, 13]. This research aimed to obtain the 3D structure of the ZIP2 and ZIP4 proteins that can be applied to further studies, especially molecular simulations.

MATERIALS AND METHODS

This research is in silico with a homology and molecular modelling approach. The databases used are National Center for Biotechnology Information (NCBI) (National Center for Biotechnology Information (nih.gov)), (Swiss Model (SWISS-MODEL (expasy.org)), and UniProt (UniProt). Validation and protein function using several sites such as Verify3D (Verify3D–UCLA-DOE Institute), ERRAT (ERRAT: An Empirical Atom-Based Method for Validating Protein Structures (1993-2015) | (ucla.edu), Procheck (PROCHECK. bio. tools), TMHMM 2.0 (TMHMM-2.0-Services-DTU Health Tech).

Nucleotide sequence determination for amino acid sequences of human ZIP2 and ZIP4

Based on the NCBI database, the DNA sequences of the ZIP2 and ZIP4 proteins were searched. The search results were obtained successively from the "gene" search with the official full name SLC39A2 solute carrier family 39 member 2 (Homo sapiens) and SLC39A4 solute carrier family 39 member 4 (Homo sapiens). Then, an Open Reading Frames (ORFs) analysis was performed on all DNA sequences from SLC39A2 and SLC39A4. Amino acid sequences translated from peptide sequence results from homology sequence tracing on NCBI. The results of ORFs with the highest similarity DNA sequences were used for BLASTp analysis from NCBI to determine the amino acid sequences of target proteins with high similarity [13].

Amino acid sequence tracing and homology modelling of human ZIP 2 and ZIP 4

After tracing the homology of the protein amino acid sequences of ZIP2 and ZIP4 with BLASTp on NCBI, the ZIP2 sequence with Reference Sequence: NP_055394.2 (Zinc transporter ZIP2 isoform a (Homo sapiens) and ZIP4 sequence with Reference Sequence: NP_001361768.1 (Zinc transporter ZIP4 isoform 4 precursors (Homo sapiens). Furthermore, the amino acid sequences of the two target proteins are stored in fasta format for protein modeling-homology modelling using Swiss Model on target proteins ZIP2 and ZIP4 on each reference sequence obtained. The model's homology results are from the highest critical scoring structure. The protein model is saved in a PDB file, which will be used for further structural analysis. The visualization used is Biovia Discovery Studio [14].

Fig. 1: The structure of Zn-carnosince

Analysis of geometrical conformation, stereochemical quality, and physicochemical properties of proteins

This study used the PROCHECK tool, ProtParam, and TMHMM database applications to determine the conformational geometry and stereochemical quality. The analysis carried out on the application is to visualize the Ramachandran diagram and physicochemical properties. In addition, this application is also used to determine the amino acid composition of the target protein, as well as other physicochemical parameters, protein expression location, topology, and transmembrane segment of the target protein.

Analysis of binding site with molecular docking

This study was done through a blind docking technique with the grid box size for ZIP2 being x, y, and z sizes of 52, 54, and 80, respectively. The grid box sizes for ZIP4 are x,y, and z sizes of 56, 72, and 100, respectively. Molecular docking used is flexible docking to determine the most stable conformation and orientation with minimal energy between Zn-carnosine and ZIP2 or ZIP4 proteins. Zn-carnosine and target protein structures were saved in. pdb and. pdbqt formats [15]. Molecular docking was performed using AutoDock Vina software with AutoDock Tools 1.5.6 (www.vina.scripps.edu). The peptide structure was visualized and prepared using MarvinSketch 20.12 (www.chemaxon.com), while the target protein structure was visualized and prepared using Biovia Discovery Studio 19.1.0 (www.3ds.com) [17]. The 2D structure of Zn-carnosine is as follows fig. 1.

RESULTS AND DISCUSSION

Searching nucleotide sequences of ZIP2 and ZIP4 using the NCBI database found that the ZIP2 target protein was in the SLC39A2 gene, and for ZIP4, the target protein was in the SLC39A4 gene. Based on the analysis of the Ensembl database, the SLC39A2 gene sequence is located on chromosome 14 in the q11.2 region (20.999.225–21.001.887). Fig. 2 shows the presence of the SLC39A2 gene. The SLC39A4 gene is located on chromosome 8 region 24.3 (144,409,742–144,416,844).

The SLC39A2 sequence had 2511 bp of nucleotides, and the SLC39A4 sequence had 4431 bp of nucleotides. They were analyzed by Open Reading Frame Finder (ORF Finder) to obtain protein translation segments using BLAST. The ORF will find several amino acid sequences translated into all nucleotide sequences SLC39A2 and SLC39A4. The longest translated amino acid values from each nucleotide sequence SLC39A2 and SLC39A4 will be used as templates for the ZIP2 and ZIP4 target proteins previously carried out by BLASTp. Table 1 shows the ORF results of the nucleotide sequences SLC39A2 and SLC39A4 with the+strand.

(a)

(b)

Fig. 2: The location of the SLC39A2 gene is on chromosome 14 (a), while SLC39A4 is on chromosome 8 (b)

Each ORF results in a nucleotide sequence; the longest amino acid is determined and followed by BLASTp analysis to determine the ZIP2 and ZIP4 target protein templates. For example, ORF finder analysis on the nucleotide SLC39A2 found that the ORF 8 region at nucleotides 1655–2287 has the longest amino acid sequence, 210 amino acids translated from 633 nucleotides. Furthermore, ORF 8 was carried out by BLASTp on NCBI to determine the highest similarity of the longest amino acid sequence in table 2. In addition, an ORF finder analysis was performed on the SLC39A4 nucleotide and translated from 678 nucleotides. Therefore, to find out the protein designated by ORF 14, it is necessary to carry out homology similarity from the NCBI database using the BLASTp tool, resulting in phylogenetic data in table 3.

Table 1: Five ORF finder results of the nucleotide sequences SLC39A2 and SLC39A4 with the longest amino acids

Label Start Stop Length
Nucleotide Amino acid
SLC39A2 ORF 8 1655 2287 633 210
ORF 11 1503 1901 399 132
ORF 2 1603 1893 291 96
ORF 7 1460 1654 195 64
ORF 4 155 304 150 49
SLC39A4 ORF 14 2628 3305 678 225
ORF 2 637 1236 600 199
ORF 7 536 970 435 144
ORF 12 1233 1622 390 129
ORF 11 3476 3823 348 115

Table 2: Five genes with the highest query cover value in ORF 8-SLC39A2

Description Max score Total score Query cover (%) E value Identn (%) Accession
Zinc transporter ZIP2 isoform a [Homo sapiens] 409 409 100 4e-145 100 NP_055394.2
Zinc transporter ZIP2 [Mus musculus] 311 311 100 2e-106 80.95 NP_001034765.2
Zinc transporter ZIP1 [Danio rerio] 160 160 96 3e-47 41.63 NP_997748.2
Zrt (ZRT), Irt-(IRT-) like Protein Transporter [Caenorhabditis elegans] 102 102 97 1e-24 33.80 NP_001122755.1
Zinc/iron-regulated transporter-related protein 42C.1 [Drosophila melanogaster] 92.8 92.8 83 4e-21 36.75 NP_525107.1

Table 3: Five genes with the highest query cover value at ORF 14-SLC39A4

Description Max score Total score Query cover (%) E value Identn (%) Accession
Zinc transporter ZIP4 isoform 4 precusor [Homo sapiens] 64.7 64.7 28 7e-11 76.81 NP_001361768.1
Zinc transporter ZIP4 precusor [Mus musculus] 61.6 61.6 20 8e-10 68.75 NP_0832340.1
Zinc transporter ZIP4 isoform 2 precusor [Homo sapiens] 64.7 64.7 28 8e-11 76.81 NP_570901.3
Zinc transporter ZIP4 isoform 1 [Homo sapiens] 64.7 64.7 28 9e-11 76.81 NP_060237.3
Zinc transporter ZIP4 isoform X1 [Homo sapiens] 63.5 63.5 20 2e-10 95.74 XP_024302957.1

The NCBI access number was obtained based on ORF and BLASTp analysis, which was used as an amino acid sequence template for molecular modelling. Based on these two analyses, the NCBI access number found that the target protein for ZIP2 was NP_055394.2 while ZIP4 was NP_001361768.1. Also, the results of modelling ZIP2 and ZIP4 on the SwissModel, are shown in table 4 and 5, respectively.

The modelling of 6pgi.1. A as the ZIP2 model and 4x82.1 as the ZIP4 model, respectively, is depicted in fig. 3 and fig. 4. Meanwhile, determining the physicochemical properties of the 3D model of the ZIP2 and ZIP4 proteins is in table 6 and table 7. The conformations of the 3D models of the ZIP2 and ZIP4 proteins are depicted in the Ramachandran diagrams in fig. 4a and 4b, respectively. ZIP2 and ZIP4 proteins are proteins located on the cell membrane. Therefore, table 8 describes the amino acid residues transmembrane in ZIP2 and ZIP4. The transmembrane residues ZIP2 and ZIP4 are also depicted in each amino acid residue's hydropathy and amphipathy profiles, forming the transmembrane properties in fig. 5 and 6.

Fig. 3: ZIP2 3D protein modelling on the 6phi.1. A. model template

Fig. 4: 3D ZIP 4 protein modelling on 4 x 8 2.1. A and 4x82.1. B. model templates

Table 4: The top five protein modelling with the swiss model on ZIP2

Template Sequence identity Sequence similarity Range Coverage QMEAN
6pgi.1. A 21.116 0.298 3-308 0.812 0.50
5tsa.1. A 20.717 0.295 3-308 0.812 0.50
6pgi.1. A 17.073 0.272 221-307 0.265 0.08
5tsa.1. A 17.500 0.276 223-307 0.259 0.08
5tsa.1. A 15.385 0.283 43-122 0.168 0.04

Table 5: The top five protein modelling with the swiss model on ZIP4

Template Sequence identity Sequence similarity Range Coverage QMEAN
4x82.1. A 69.39 0.51 34-229 0.35 0.22
4x82.1. B 69.39 0.51 34-229 0.35 0.21
6pgi.1. A 18.90 0.29 229-547 0.46 0.29
5tsa.1. A 18.97 0.30 230-547 0.46 0.29
4x82.1. A 68.82 0.51 63-232 0.31 0.16

Table 6: Physicochemical properties of the 3D ZIP2 model on the 6pgi.1. A. model template

Physicochemical identities 3D ZIP2 model Amino acid composition No. Composition (%)
Number of amino acids 309 Ala (A) 27 12.3
Molecular weight 32742.38 Arg (R) 19 3.2
Theoretical pI 5.85 Asn (N) 16 0.6
No. of negatively charged (Asp+Glu) 23 Asp (D) 36 1.6
No. of positively charged (Arg+Lys) 16 Cys (C) 10 2.6
Atomic composition Gln (Q) 24 3.2
Carbon (C) 1501 Glu (E) 35 5.8
Hydorgen (H) 2340 Gly (G) 21 11.3
Nitrogen (N) 384 His (H) 14 3.6
Oxygen (O) 404 Ile (I) 35 3.9
Sulfur (S) 15 Leu (L) 52 16.5
Formula C1501H2340N384O404S16 Lys (K) 39 1.9
Total number of atoms 4645 Met (M) 17 2.6
Aliphatic index 112.46 Phe (F) 27 6.8
Grand average of hydropathicity 0.683 Pro (P) 25 3.6
Instability index 41.17 Ser (S) 39 7.4
Thr (T) 26 3.2
Trp (W) 1 1.6
Tyr (Y) 18 1.0
Val (V) 24 7.1

Human zinc transporters ZIP2 and ZIP4 are two of the 14 types of the ZIP family, both of which are types of transport proteins responsible for the transport of mineral zinc from extracellular to intracellular. Therefore, ZIP2 and ZIP4 protein modelling are needed to determine the 3D structure of the protein, its function, and the protein's physicochemical properties. Furthermore, protein modelling can be used in determining the interaction of receptors to ligands in determining drug candidates and their role in molecular dynamics simulations [16].

The 3D structures deposited in the Protein Data Bank (PDB) database are not always complete. Therefore, it is necessary to approach protein modelling with homology modelling to determine the comparative model of the protein we need. The modelling in this research uses the Swiss model to determine the protein homology of human ZIP2 and ZIP4. Therefore, several databases are needed from the nucleotide sequences that will be translated into amino acid sequences to determine the tertiary structures of the protein to obtain the 3D structure of human ZIP2 and ZIP4.

Table 7: Physicochemical properties of the 3D ZIP4 model on the 4x82.1. A and 4x82.1. B. model templates

Physicochemical identities 3D ZIP4 Model Amino acid composition No. Composition (%)
Number of amino acids 582 Ala (A) 72 12.4
Molecular weight 61642.95 Arg (R) 21 3.6
Theoretical pI 5.24 Asn (N) 9 1.5
No. of negatively charged (Asp+Glu) 52 Asp (D) 22 3.8
No. of positively charged (Arg+Lys) 27 Cys (C) 14 2.4
Atomic composition Gln (Q) 21 3.6
Carbon (C) 2776 Glu (E) 30 5.2
Hydrogen (H) 4356 Gly (G) 48 8.2
Nitrogen (N) 736 His (H) 23 4.0
Oxygen (O) 804 Ile (I) 8 1.4
Sulfur (S) 23 Leu (L) 107 18.4
Formula C2776H4356N736O804S23 Lys (K) 6 1.0
Total number of atoms 8695 Met (M) 9 1.5
Aliphatic index 109.36 Phe (F) 21 3.6
Grand average of hydropathicity 0.373 Pro (P) 35 6.0
Instability index 46.8 Ser (S) 53 9.1
Thr (T) 24 4.1
Trp (W) 9 1.5
Tyr (Y) 10 1.7
Val (V) 40 6.9

Starting from the search for the nucleotide level database, the nucleotide sequences known that the chromosomes in the SLC39A2 and SLC39A4 genes are genes encoding the protein ZIP2 and ZIP4. Furthermore, based on the ORF analysis of the nucleotide sequences, it is known that the ZIP2 protein has homology in the "zinc transporter ZIP2 isoform a [Homo sapiens]" protein database in NCBI with a 3D protein structure template homology in PDB "6pgi.1. A". On the other hand, an ORF analysis of nucleotide sequences shows that the ZIP4 protein is like "zinc transporter ZIP4 isoform 4 precursors [Homo sapiens]" in the NCBI database, which has a 3D structure called "4x82.1. A" that is like the structure of the homology template.

Fig. 3 and fig. 4 are the 3D protein structures of ZIP2 and ZIP4 with physicochemical properties in table 6 and table 7, and it is known that in ZIP2 protein, there are 309 amino acids, the theoretical pI is 5.85 with leucine as the largest constituent amino acid, which is 16.5%, followed by Alanine 12.5% and the third highest was Glysin at 11.3%. In the 3D structure of the ZIP4 protein, it is known that there are 582 amino acids, with a theoretical pI value of 5.24. Like protein ZIP2, Protein ZIP4 contains much leucine with a composition of 18.4%, then Alanine at 12.4%, and Serine at 9.1%. The Ramachandran diagram (fig. 5) shows a 3D model of the ZIP2 and ZIP4 proteins, which have more than 85% of the amino acid residues distributed in the quadrant 1 conformation, where the stereochemistry formed in the secondary structure of the protein is antiparallel-sheet and parallel-sheet. Meanwhile, in fig. 5a and 5b, other amino acid residues are in quadrants 2 and 3 as amino acids, forming the secondary structures of the left-handed and right-handed helix. Less than 1% of ZIP2 and ZIP4 proteins are in quadrant 4, where the smaller the percentage of amino acid residues in quadrant 4, the more stable the protein model is formed. The stability of protein models is important in preparing proteins for molecular simulations in bioinformatics applications such as molecular docking and molecular dynamics.

Fig. 5: Ramachandran diagram on 3D models ZIP2 (a) and ZIP4 (b)

Table 8: Topological locations of transmembrane sequences on ZIP2 and ZIP4

ZIP2 protein Transmembrane location ZIP4 protein Transmembrane location
TMS 1 9-29 TMS 1 327-249
TMS 2 47-67 TMS 2 355-375
TMS 3 107-127 TMS 3 559-582
TMS 4 165-185 TMS 4 588-611
TMS 5 193-213 TMS 5 619-641
TMS 6 225-246
TMS 7 256-276
TMS 8 288-308

The ZIP2 and ZIP4 proteins are also distributed in the cell membrane as zinc mineral transporter receptors, so analyzing the secondary structure of amino acids at the transmembrane position is necessary. Based on the hydropathy and amphipathy analysis of amino acid residues in the ZIP2 and ZIP4 proteins, there are 8 secondary structures and 5 secondary structures at the transmembrane position, respectively, described in table 8. Analysis of transmembrane proteins is important in determining zinc minerals' binding and transport mechanisms in the extracellular transport process. to intracellular and vice versa. More than half of the ZIP transporters have His-rich sequences found in the cytoplasmic part of the intracellular loop between TMDs III and IV [17]. The presence of this loop has its role in the ZIP transporter family. In ZIP 1, this loop plays a role in the di-leucine sorting signal as a mediator of endocytosis.

On the other hand, this loop in ZIP 4 plays a role in mediating ubiquitination and degradation [18]. In mammals, in the LIV-1 subfamily, there is a metalloprotease motif (HEXPHEXGD) on TMD V and a CPALLY motif located just before TMD 1, except for ZIP 7 and 13 [19]. The location of ZIP transporters is mostly on the plasma membrane. However, ZIP and ZnT transporters also change with zinc stimulation and availability in the subcellular. The diverse localization and transport of zinc are important in homeostasis and biological processes [20-22].

(a) (b)

Fig. 6: Interaction Zn-carnosine with ZIP2 (a) and ZIP4 (b) through molecular docking simulations

Zn-carnosine (C9H13N4O3Zn) is a zinc-binding peptide, a third-generation zinc supplement. Carnosine, a peptide binding with zinc, enhances cell bioabsorption and bioavailability [23-25]. To determine the binding activity of ZIP2 and ZIP4 with zinc supplements, Zn-carnosine is used as a ligand to determine the binding site on ZIP2 and ZIP4. Fig. 6a and 6b are the conformation and orientation of Zn-carnosine binding with ZIP2 and ZIP4. The results obtained several amino acids on ZIP2 formed hydrogen bonds with Zn-carnosine, such as Glu281 formed hydrogen bonds along 2.32 A, His216 along 2.84A, and Ser284 along 3.23. Another amino acid in ZIP2 that forms van der Waals Zn-carnosine bonds is Arg46. Meanwhile, fig. 6b shows the hydrogen bond that is the interaction of amino acids in ZIP4 with Zn-Carnosine, while the hydrogen bond occurs at Ala138 along 2.84 A, His197 along 2.99, Gln154 along 3.06, Thr155 along 2.67, and Gln148 along 3.05. Lys157 on ZIP4 forms van der Waals bond with Zn-Carnosine.

Molecular docking simulation shows the binding side of ZIP2 and ZIP4 on amino acids that interact with hydrogen and van der Waals bonds. The binding amino acids on ZIP2 include Glu281, His216, Ser284, and Arg46. The binding amino acids in ZIP4 include Gln148, Gln154, Thr155, His197, Ala138, and Lys157. Based on the analysis of transmembrane proteins (TMS) in table 8, in ZIP2, the amino acids Arg46, His216, Glus281, and Ser 284 are not in the transmembrane region, which is in the loop part of the ZIP2 protein both in the interior and exterior of the protein. The amino acids in ZIP4 are not in the TMS region but in the exterior and interior loops of the protein. The factor of the region causes a potential interaction on Zn-carnosine.

CONCLUSION

The 3D structures of the ZIP2 and ZIP4 proteins have been obtained by tracing the nucleotide sequences encoding the genes SLC39A2 and SLC39A4. These two genes are translated into amino acid-forming proteins in ZIP2 and ZIP4. Based on the physicochemical properties of the 3D structure of the ZIP2 and ZIP4 proteins, each comprises 309 amino acids and 582 amino acids with pI values of 5.85 and 5.24. The amino acid composition analysis showed that both proteins contain many Leucine amino acids. The Ramachandran diagram concludes that both proteins are stable in the stereochemical conformation forming a secondary structure. Therefore, they suit molecular simulation applications, such as molecular docking and molecular dynamics. Its function is to move zinc minerals across cell membranes. ZIP2 protein comprises 8 secondary structures of a transmembrane protein, while ZIP4 protein comprises 5 secondary structures. Molecular docking simulation shows the binding side of ZIP2 and ZIP4 on amino acids that interact with hydrogen and van der Waals bonds with Zn-carnosine. The binding amino acids on ZIP2 include Glu281, His216, Ser284, and Arg46. The binding amino acids in ZIP4 include Gln148, Gln154, Thr155, His197, Ala138, and Lys157.

ACKNOWLEDGMENT

Research Organization for Health by “Vaccine and Drugs Research Program”-The National Research and Innovation Agency, Republic of Indonesia fiscal year 2023.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

GS worked all experiments and technical design, ML and FF the main conceptual ideas and proofreader, MYP and FF designed the experiments and proofreader; NG edited the manuscript and proofreader. GS proposed the experiment in discussions with ML, MYP, and FF. GS wrote the manuscript.

CONFLICT OF INTERESTS

The authors declare that there are no conflicts of interest in this article.

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