Investigating the ATP binding pocket of CX3CL1 binding protein 2 using in silico approach

Investigating the ATP binding pocket of CX3CL1 binding protein 2 using in silico approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Investigating the ATP binding pocket of CX3CL1 binding protein 2 using in silico approach Rimjhim Kumari, Satinder Kaur, Rachna Hora, Prakash Chandra Mishra This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2985578/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2024 Read the published version in Journal of Proteins and Proteomics → Version 1 posted 7 You are reading this latest preprint version Abstract Plasmodium falciparum (Pf) causes the most fatal form of malaria owing to its ability to cytoadhere in the microvasculature of various organs in the body. In addition to the Pf erythrocyte membrane protein 1 (PfEMP1) family that binds diverse host receptors, CX3CL1 binding proteins 1 and 2 (CBP1 and 2) also bind the endothelial chemokine ‘CX3CL1’ to effect cytoadhesion of parasite infected erthrocytes. CBP2 is a multifaceted protein that binds nucleic acids, Pf skeleton binding protein (PfSBP1) and ATP. ATP binding to the cytoplasmic domain of CBP2 (cCBP2) induces structural changes in the protein, and hints at its role in cell signaling. In this study, we have attempted to identify the ATP binding pocket of CBP2 using an in silico approach. We have also delineated the type of interactions and amino acid residues that are likely to bind ATP. As CX3CL1 binding proteins are central to parasite biology, the obtained information is likely to form the basis for inhibitor and drug design against this molecule. Malaria Plasmodium falciparum CBP2 CX3CL1 ATP Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plasmodium falciparum (Pf) is responsible for causing the most fatal form of malaria in humans [ 1 ]. Asexual multiplication of this pathogen occurs inside infected RBCs (iRBCs) [ 2 ] where it exports a variety of proteins to the parasitized host cell. These include families like Pf erythrocyte membrane protein 1 (PfEMP1), Rifin, Stevor, Surfin etc. collectively termed the ‘malaria exportome’ [ 3 ]. Proteins of the exportome have diverse roles in altering iRBC rigidity and adhesiveness, their ability to uptake nutrients and escape the host immune response [ 4 ]. Cytoadherence, i.e. adhesion of parasitized erythrocytes with host endothelial cells in tissue microvasculature is chiefly mediated by the exported PfEMP1 family [ 5 ] that is reported to bind a variety of host endothelial receptors viz. CD36, ICAM-1, VCAM, CSA etc. in different tissues [ 6 ]. In 2003, Hatabu et. al. had identified the chemokine ‘CX3CL1’ as another receptor located on host endothelial membrane that is involved in cytoadhesion of parasitized RBCs (pRBCs) in the brain of cerebral malaria patients[ 7 ]. Constitutive expression of CX3CL1 (also called fractalkine) is seen in a variety of non-hematopoietic tissues including brain, heart, lungs and kidneys both as membrane bound and soluble isoforms [ 8 ], [ 9 ]. While the membrane-bound form of fractalkine (FKN) is an adhesion molecule, its soluble form chemoattracts monocytes and natural killer (NK) cells [ 10 ], [ 11 ]. A P. falciparum protein family 'hypothetical 8' identified by Sargeant et al [ 12 ] has two members that are expressed on the surface of iRBCs which act as parasite ligands involved in CX3CL1 mediated cytoadherence [ 13 ]. These are CX3CL1 binding proteins 1 (CBP1; PF3D7_0113900) and 2 (CBP2; PF3D7_1301700), which share 32% sequence identity. CBP2 is a PEXEL positive multi-transmembrane protein that contains a nuclear localization signal as well as a cold shock DNA binding domain. Its PEXEL motif is likely to be cleaved during export leading to formation of a 2TM (two transmembrane) protein product that localizes at iRBC membranes [ 14 ]. CBP2 is also reported to co-localize with Pf skeleton binding protein (PfSBP1) at Maurer's clefts (MCs) during the ring stage of parasite development [ 15 ]. MCs are specialized membranous structures within iRBC cytosol that are formed by the asexual parasites during their early ring stage [ 16 ], [ 17 ]. It is believed that PfSBP1 resides in Maurer's clefts and works with other proteins like MAHRP1, Pf322, REX1, and REX2 to transport PfEMP1[ 18 ]. The cytoplasmic domain of CBP2 (cCBP2) is also known to directly bind PfSBP1, nucleic acids (DNA/RNA) and ATP [ 19 ]. Furthermore, both PfSBP1 and CBP2 have been found to be present in extracellular vesicles (EVs) produced by parasitized RBCs. EVs are responsible for transport of packaged proteins and nucleic acids to target cells and play roles in gametocyte development, inter-parasite communication and modulation of the host immunological response [ 20 ]–[ 24 ]. ATP binding of 2TM protein ‘CBP2’ is suggestive of its role as a purinergic receptor that effects cellular signaling. Binding of cCBP2 with ATP induced structural changes in the protein leading to its oligomerization [ 19 ]. ATP binding negatively impacted the interaction between cCBP2 and PfSBP1. While NMR studies identified ATP atoms H2 and H8 from adenine, and H1 of its sugar to be involved in cCBP2 binding, no information is available on the interacting residues from CBP2. Here, we have used in silico approach including molecular docking to identify residues from CBP2 which are likely to be engaged in interaction with ATP. Considering the importance of ATP binding sites on various proteins as valuable drug targets[ 25 ], this information may form the foundation for drug designing against CBP2. Materials and Methods Protein sequence retrieval and ATP Binding Site Prediction The sequence of full length CBP2 was retrieved from PlasmoDB (PF3D7_1301700). ATP binding site on CBP2 was predicted by using ATPint [ 26 ]. ATPint predicts the ATP-interacting residues on a protein with Support Vector Machine (SVM) by using the sequence of the protein. The amino acid sequence (in FASTA format) of CBP2 was used as the input for prediction. Retrieval of structures and ATP binding site prediction The three dimensional (3D) structure of CBP2 (PF3D7_1301700) was obtained from AlphaFold [ 27 ] via PlasmoDB [ 28 ]. Its ATP binding residues were predicted by using ATPbind [ 29 ], which is an SVM based tool that identifies ATP binding pockets based on the protein three dimensional (3D) structure. Molecular docking and interaction analysis The 3D protein structure was prepared for docking by adding hydrogens and charges using UCSF Chimera 1.16 [ 30 ]. The structure of ATP was retrieved from https://pubchem.ncbi.nlm.nih.gov (PubChem CID − 5957).The 3D structure of ATP was downloaded in SDF format and further used for docking studies. Molecular Docking was performed using Autodock via CHIMERA [ 30 ]. Both, CBP2 as receptor and ATP as ligand were prepared using CHIMERA in PDBQT format. Based on predictions from ATPint & ATPbind and information based on our report of ATP binding to recombinant cCBP2 [ 19 ], a grid box was selected to perform the docking. Others parameters for ligand and receptor were set to default [ 31 ]. After docking, the result was saved in PDBQT format. The different generated complexes were used for further analysis using LigPlot + and Prodigy [ 32 ]. Results and Discussion Sequence based ATP binding site prediction P. falciparum CBP2 may be essential for parasite biology as multiple deletion attempts of its gene were unsuccessful [ 15 ], [ 33 ]. However, the genome wide piggyBac transposon approach showed that its gene could be mutated [ 34 ]. Keeping in mind the importance of CBP2 in a central pathogenic mechanism i.e. cytoadherence and ATP binding ability, we analysed the sequence and structure of PfCBP2 (PF3D7_1301700). Its full length sequence was downloaded from PlasmoDB and analysed using ATPint. Since the cytoplasmic domain of CBP2 spanning residues 30–160 has earlier been reported to bind ATP experimentally [ 19 ], we considered only this region for our evaluation. Arg43, Pro64, Val70, Tyr105, Arg117, Lys143, Asn 146, Lys152 and Val160 were predicted to interact with ATP. Structure based ATP binding site prediction: The predicted structure of full length CBP2 is available on Alpha Fold (Fig. 1 a). This 3D structure was verified for its structural integrity using Ramachandran plot[ 35 ] (Figure S1). All the residues of the predicted structure were found in the favoured or allowed regions. This 3D structure was used for prediction of ATP binding residues using ATPbind where structural coordinates are used as input. Here, Arg43 and Lys137 were predicted to interact with CBP2. Molecular docking and analysis: Since ATPint and ATPbind results showed different sites for ATP binding, we used molecular docking for understanding ATP interaction with CBP2. Molecular docking was performed by Autodock using structure of ATP ligand downloaded from PubChem (CID 5957) (Fig. 1 b). The CBP2 structure and ligand were prepared using Chimera for docking by generating a grid box (Fig. 2 & Table S1). The grid box was selected for the docking experiment based on the result obtained from ATPint & ATPbind and previous binding report of ATP with cytoplasmic domain of CBP2 (cCBP2; residues 30–160 of full length CBP2 [ 19 ]. Nine probable complexes between CBP2 and ATP were generated by docking. PDB file of complexes generated from docking results were used for interaction studies using Ligplot+. Binding energies of the complexes were calculated using Prodigy (Table S2) [ 36 ]. The binding energies of all the complexes were found to be very similar ranging from − 5.62 to -5.23 kCal/mol and docking score ranging between − 7.189 to -6.138. In the complex I which had highest docking score of -7.189, ATP was seen to nestle into a deep binding pocket with adenine forming contacts with residues lying deeper in the cavity and the phosphates interacting with the more exterior amino acids (Fig. 3 ). While complex 1 showed all the three interactions reported by NMR studies (H1 of ribose sugar; H2 & H8 of adenine from ATP) [ 19 ], rest of the other complexes showed contacts involving either one or two of these atoms. Therefore, we selected complex 1 for detailed analysis. Carbons neighboring each of the above ATP atoms were seen to be engaged in hydrophobic interactions (C1 of ribose with Arg115, C2 of adenine with Ser38 & Ile42 and C8 of adenine with Asn35) (Table 1 and Fig. 4 a). O atoms of different phosphate groups of ATP were seen to form hydrogen bonds. Specifically, P1 formed hydrogen bonds with Gln114, P2 with His145, Asn146 & Tyr105 and P3 with Tyr105, Lys108 & Val106 (Fig. 4 b). Additional observed interactions are listed in Table 1 . Table 1 Table 1 Summary of interactions in complex 1 for CBP2-ATP interaction . Table lists various hydrogen bonds and hydrophobic interactions between the two binding partners. Hydrogen bond Hydrophobic bond ATP CBP2 ATP CBP2 O of P1 Gln114 C1(Ribose) Arg115 O of P2 His145,Asn146,Tyr105 C2(Adenine) Ser38,Ile42 O of P3 Val106,Lys108, Tyr105 C8(Adenine) Asn35 N3(Adenine) Arg115 N6(Adenine) Ala150 N6(Adenine) Asp147 O of P1 Gly111,Ser110 C2(Ribose) Asp34 Overall, we have identified amino acid residues of CBP2 that are likely to interact with ATP by using an in silico approach. Our study may therefore form the basis for structure based inhibitor design against an important parasite molecule ‘CBP2’. Site directed mutagenesis of these residues can be used to confirm the importance of each of these residues in ATP binding. Declarations Acknowledgments RK is receiving scholarship from DBT, Government of India. SK is a DBT-SRF. The laboratories of PCM and RH were funded by DBT, RUSA and DST, Government of India. The authors have no relevant financial or non-financial interests to disclose. The authors declare that there are no conflicts of interest. 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Supplementary Files Supplementaryfigureandtables.docx Cite Share Download PDF Status: Published Journal Publication published 26 Mar, 2024 Read the published version in Journal of Proteins and Proteomics → Version 1 posted Editorial decision: Revision requested 25 Dec, 2023 Reviews received at journal 24 Dec, 2023 Reviewers agreed at journal 14 Jun, 2023 Reviewers invited by journal 14 Jun, 2023 Editor assigned by journal 29 May, 2023 Submission checks completed at journal 26 May, 2023 First submitted to journal 26 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2985578","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":216487172,"identity":"f7025779-e958-4332-b693-09efb8495f91","order_by":0,"name":"Rimjhim Kumari","email":"","orcid":"","institution":"Guru Nanak Dev University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rimjhim","middleName":"","lastName":"Kumari","suffix":""},{"id":216487173,"identity":"b08e13ce-c36e-44c1-9db6-a452ce2c8c52","order_by":1,"name":"Satinder Kaur","email":"","orcid":"","institution":"Guru Nanak Dev University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Satinder","middleName":"","lastName":"Kaur","suffix":""},{"id":216487174,"identity":"29fe43f8-cf48-459a-8f6e-b8d98ae7734c","order_by":2,"name":"Rachna Hora","email":"","orcid":"","institution":"Guru Nanak Dev University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rachna","middleName":"","lastName":"Hora","suffix":""},{"id":216487175,"identity":"ffe1f562-6b07-4d51-928a-5b29a28b20c7","order_by":3,"name":"Prakash Chandra Mishra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYHACNoYEECXBfADEBoMDRGgxAGphSyBBCwNYC48BXAteYC52gO3Bg4o/cvyzez6/Lig7zMDffoDxcAEeLZazE9gNEs4YGEvcObvNesa5wwwSZxIYDs/Ao8XgdgKbRGKbQeIGidxtxrxthxkYbjAwHOYhQkv9BomcZ2At8sRqSTCQyGF+DNJiQEiL5ezEdqBfjA1n3EgzY+Y5l85jeCaxAa8Wc+nkYw9/VMjJ889IfvyZp8xaTu744cOf8TqMgbEBxmaTABJAxQgRHFoQgPkDXqWjYBSMglEwYgEAirRKL/0k8vwAAAAASUVORK5CYII=","orcid":"","institution":"Guru Nanak Dev University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Prakash","middleName":"Chandra","lastName":"Mishra","suffix":""}],"badges":[],"createdAt":"2023-05-26 12:14:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2985578/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2985578/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s42485-024-00133-z","type":"published","date":"2024-03-26T09:28:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":42147334,"identity":"ab65b95f-1eb6-40c4-aaa9-0433e2473929","added_by":"auto","created_at":"2023-08-25 16:52:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":286473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eStructures of CBP2 and ATP.\u003c/em\u003ea) Cartoon representation of full length CBP2 obtained from Alpha fold: Cytoplasmic domain of CBP2 (cCBP2) known to bind ATP (30-160) is shown in cyan. Rest of the protein is pink. b) Structure of ATP obtained from PubChem (CID – 5957). Adenine, ribose sugar and phosphates are labeled. Carbon atoms are shown in beige, nitrogen in blue, oxygen in red, phosphorus in orange and hydrogen in white. Atoms in adenine (N1 to N9), ribose (C1 to C5) and triphosphate (P1 to P3) are separately numbered.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2985578/v1/0257a6b234d49aae9f5f5e4c.png"},{"id":42149427,"identity":"c9c48ff5-fa97-4563-b67a-bb7571c54461","added_by":"auto","created_at":"2023-08-25 17:08:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":291498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCartoon representation of CBP2 in chimera showing the grid box and docked ATP.\u003c/em\u003e Inset shows selected coordinates for grid box.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2985578/v1/c5302d8be7bbacfee0255ef0.png"},{"id":42147331,"identity":"3d1e3d69-7219-46c8-ba84-e32265a1297c","added_by":"auto","created_at":"2023-08-25 16:52:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":248961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMolecular surface view of complex 1 showing CBP2 and docked ATP.\u003c/em\u003e Molecular surface view of CBP2 protein is shown in light blue. ATP (red sticks) are seen to bind in a deep pocket on the protein.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2985578/v1/8fdf750a0c89ffd82e715dd6.png"},{"id":42148393,"identity":"7f090b8e-c8be-4844-b08b-3cf8709f6a70","added_by":"auto","created_at":"2023-08-25 17:00:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":257999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLigPlot+ analysis of complex 1\u003c/em\u003e. ATP is shown in the centre (ball and stick representation). Amino acid residues of CBP2 involved in interactions are shown either as ball and stick (residues forming hydrogen bonds) or as arcs (residues showing hydrophobic interactions). a) Most significant hydrophobic interactions are shown as red dotted lines. Additional residues involved in hydrophobic binding are shown with emanating rays. b) Hydrogen bonds are shown as green dotted lines. Bond length is labelled. Arg115 (shown as ball and stick) forms both hydrophobic interactions and hydrogen bonds (a \u0026amp; b).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2985578/v1/431c4ac36d31b773cfab37bd.png"},{"id":58486606,"identity":"e3a0afa5-f351-40bf-8886-9c97d914c6dd","added_by":"auto","created_at":"2024-06-17 09:28:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1611211,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2985578/v1/95a42985-a2ad-474a-b39b-c1b6e16698b6.pdf"},{"id":42147336,"identity":"6b2aa4c9-5933-4772-bff9-de483250c937","added_by":"auto","created_at":"2023-08-25 16:52:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":155194,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigureandtables.docx","url":"https://assets-eu.researchsquare.com/files/rs-2985578/v1/7c5cda0991b6fbd0d011656a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigating the ATP binding pocket of CX3CL1 binding protein 2 using in silico approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (Pf) is responsible for causing the most fatal form of malaria in humans [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Asexual multiplication of this pathogen occurs inside infected RBCs (iRBCs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] where it exports a variety of proteins to the parasitized host cell. These include families like Pf erythrocyte membrane protein 1 (PfEMP1), Rifin, Stevor, Surfin etc. collectively termed the \u0026lsquo;malaria exportome\u0026rsquo; [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Proteins of the exportome have diverse roles in altering iRBC rigidity and adhesiveness, their ability to uptake nutrients and escape the host immune response [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cytoadherence, i.e. adhesion of parasitized erythrocytes with host endothelial cells in tissue microvasculature is chiefly mediated by the exported PfEMP1 family [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] that is reported to bind a variety of host endothelial receptors viz. CD36, ICAM-1, VCAM, CSA etc. in different tissues [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In 2003, Hatabu \u003cem\u003eet. al.\u003c/em\u003e had identified the chemokine \u0026lsquo;CX3CL1\u0026rsquo; as another receptor located on host endothelial membrane that is involved in cytoadhesion of parasitized RBCs (pRBCs) in the brain of cerebral malaria patients[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConstitutive expression of CX3CL1 (also called fractalkine) is seen in a variety of non-hematopoietic tissues including brain, heart, lungs and kidneys both as membrane bound and soluble isoforms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. While the membrane-bound form of fractalkine (FKN) is an adhesion molecule, its soluble form chemoattracts monocytes and natural killer (NK) cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A \u003cem\u003eP. falciparum\u003c/em\u003e protein family 'hypothetical 8' identified by Sargeant \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] has two members that are expressed on the surface of iRBCs which act as parasite ligands involved in CX3CL1 mediated cytoadherence [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These are CX3CL1 binding proteins 1 (CBP1; PF3D7_0113900) and 2 (CBP2; PF3D7_1301700), which share 32% sequence identity.\u003c/p\u003e \u003cp\u003eCBP2 is a PEXEL positive multi-transmembrane protein that contains a nuclear localization signal as well as a cold shock DNA binding domain. Its PEXEL motif is likely to be cleaved during export leading to formation of a 2TM (two transmembrane) protein product that localizes at iRBC membranes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CBP2 is also reported to co-localize with Pf skeleton binding protein (PfSBP1) at Maurer's clefts (MCs) during the ring stage of parasite development [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. MCs are specialized membranous structures within iRBC cytosol that are formed by the asexual parasites during their early ring stage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is believed that PfSBP1 resides in Maurer's clefts and works with other proteins like MAHRP1, Pf322, REX1, and REX2 to transport PfEMP1[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The cytoplasmic domain of CBP2 (cCBP2) is also known to directly bind PfSBP1, nucleic acids (DNA/RNA) and ATP [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Furthermore, both PfSBP1 and CBP2 have been found to be present in extracellular vesicles (EVs) produced by parasitized RBCs. EVs are responsible for transport of packaged proteins and nucleic acids to target cells and play roles in gametocyte development, inter-parasite communication and modulation of the host immunological response [\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eATP binding of 2TM protein \u0026lsquo;CBP2\u0026rsquo; is suggestive of its role as a purinergic receptor that effects cellular signaling. Binding of cCBP2 with ATP induced structural changes in the protein leading to its oligomerization [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. ATP binding negatively impacted the interaction between cCBP2 and PfSBP1. While NMR studies identified ATP atoms H2 and H8 from adenine, and H1 of its sugar to be involved in cCBP2 binding, no information is available on the interacting residues from CBP2. Here, we have used \u003cem\u003ein silico\u003c/em\u003e approach including molecular docking to identify residues from CBP2 which are likely to be engaged in interaction with ATP. Considering the importance of ATP binding sites on various proteins as valuable drug targets[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], this information may form the foundation for drug designing against CBP2.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eProtein sequence retrieval and ATP Binding Site Prediction\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe sequence of full length CBP2 was retrieved from PlasmoDB (PF3D7_1301700). ATP binding site on CBP2 was predicted by using ATPint [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. ATPint predicts the ATP-interacting residues on a protein with Support Vector Machine (SVM) by using the sequence of the protein. The amino acid sequence (in FASTA format) of CBP2 was used as the input for prediction.\u003c/p\u003e\n\u003ch3\u003eRetrieval of structures and ATP binding site prediction\u003c/h3\u003e\n\u003cp\u003eThe three dimensional (3D) structure of CBP2 (PF3D7_1301700) was obtained from AlphaFold [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] via PlasmoDB [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Its ATP binding residues were predicted by using ATPbind [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which is an SVM based tool that identifies ATP binding pockets based on the protein three dimensional (3D) structure.\u003c/p\u003e\n\u003ch3\u003eMolecular docking and interaction analysis\u003c/h3\u003e\n\u003cp\u003eThe 3D protein structure was prepared for docking by adding hydrogens and charges using UCSF Chimera 1.16 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The structure of ATP was retrieved from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (PubChem CID \u0026minus;\u0026thinsp;5957).The 3D structure of ATP was downloaded in SDF format and further used for docking studies.\u003c/p\u003e \u003cp\u003eMolecular Docking was performed using Autodock via CHIMERA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Both, CBP2 as receptor and ATP as ligand were prepared using CHIMERA in PDBQT format. Based on predictions from ATPint \u0026amp; ATPbind and information based on our report of ATP binding to recombinant cCBP2 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], a grid box was selected to perform the docking. Others parameters for ligand and receptor were set to default [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. After docking, the result was saved in PDBQT format. The different generated complexes were used for further analysis using LigPlot\u0026thinsp;+\u0026thinsp;and Prodigy [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eSequence based ATP binding site prediction\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eP. falciparum\u003c/em\u003e CBP2 may be essential for parasite biology as multiple deletion attempts of its gene were unsuccessful [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, the genome wide \u003cem\u003epiggyBac\u003c/em\u003e transposon approach showed that its gene could be mutated [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Keeping in mind the importance of CBP2 in a central pathogenic mechanism i.e. cytoadherence and ATP binding ability, we analysed the sequence and structure of PfCBP2 (PF3D7_1301700). Its full length sequence was downloaded from PlasmoDB and analysed using ATPint. Since the cytoplasmic domain of CBP2 spanning residues 30\u0026ndash;160 has earlier been reported to bind ATP experimentally [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], we considered only this region for our evaluation. Arg43, Pro64, Val70, Tyr105, Arg117, Lys143, Asn 146, Lys152 and Val160 were predicted to interact with ATP.\u003c/p\u003e\n\u003ch3\u003eStructure based ATP binding site prediction:\u003c/h3\u003e\n\u003cp\u003eThe predicted structure of full length CBP2 is available on Alpha Fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This 3D structure was verified for its structural integrity using Ramachandran plot[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (Figure S1). All the residues of the predicted structure were found in the favoured or allowed regions. This 3D structure was used for prediction of ATP binding residues using ATPbind where structural coordinates are used as input. Here, Arg43 and Lys137 were predicted to interact with CBP2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMolecular docking and analysis:\u003c/h3\u003e\n\u003cp\u003eSince ATPint and ATPbind results showed different sites for ATP binding, we used molecular docking for understanding ATP interaction with CBP2. Molecular docking was performed by Autodock using structure of ATP ligand downloaded from PubChem (CID 5957) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The CBP2 structure and ligand were prepared using Chimera for docking by generating a grid box (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; Table S1). The grid box was selected for the docking experiment based on the result obtained from ATPint \u0026amp; ATPbind and previous binding report of ATP with cytoplasmic domain of CBP2 (cCBP2; residues 30\u0026ndash;160 of full length CBP2 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nine probable complexes between CBP2 and ATP were generated by docking. PDB file of complexes generated from docking results were used for interaction studies using Ligplot+. Binding energies of the complexes were calculated using Prodigy (Table S2) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The binding energies of all the complexes were found to be very similar ranging from \u0026minus;\u0026thinsp;5.62 to -5.23 kCal/mol and docking score ranging between \u0026minus;\u0026thinsp;7.189 to -6.138. In the complex I which had highest docking score of -7.189, ATP was seen to nestle into a deep binding pocket with adenine forming contacts with residues lying deeper in the cavity and the phosphates interacting with the more exterior amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile complex 1 showed all the three interactions reported by NMR studies (H1 of ribose sugar; H2 \u0026amp; H8 of adenine from ATP) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], rest of the other complexes showed contacts involving either one or two of these atoms. Therefore, we selected complex 1 for detailed analysis. Carbons neighboring each of the above ATP atoms were seen to be engaged in hydrophobic interactions (C1 of ribose with Arg115, C2 of adenine with Ser38 \u0026amp; Ile42 and C8 of adenine with Asn35) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). O atoms of different phosphate groups of ATP were seen to form hydrogen bonds. Specifically, P1 formed hydrogen bonds with Gln114, P2 with His145, Asn146 \u0026amp; Tyr105 and P3 with Tyr105, Lys108 \u0026amp; Val106 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Additional observed interactions are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eSummary of interactions in complex 1 for CBP2-ATP interaction\u003c/em\u003e. Table lists various hydrogen bonds and hydrophobic interactions between the two binding partners.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHydrogen bond\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eHydrophobic bond\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCBP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCBP2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO of P1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGln114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC1(Ribose)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eArg115\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO of P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHis145,Asn146,Tyr105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC2(Adenine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSer38,Ile42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eO of P3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVal106,Lys108, Tyr105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC8(Adenine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAsn35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN3(Adenine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArg115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN6(Adenine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAla150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN6(Adenine)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsp147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eO of P1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGly111,Ser110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC2(Ribose)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsp34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003eOverall, we have identified amino acid residues of CBP2 that are likely to interact with ATP by using an \u003cem\u003ein silico\u003c/em\u003e approach. Our study may therefore form the basis for structure based inhibitor design against an important parasite molecule \u0026lsquo;CBP2\u0026rsquo;. Site directed mutagenesis of these residues can be used to confirm the importance of each of these residues in ATP binding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRK is receiving scholarship from DBT, Government of India. SK is a DBT-SRF. The laboratories of PCM and RH were funded by DBT, RUSA and DST, Government of India.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. F. Cowman, J. Healer, D. Marapana, and K. Marsh, \u0026ldquo;Malaria: Biology and Disease,\u0026rdquo; \u003cem\u003eCell\u003c/em\u003e, vol. 167, no. 3, pp. 610\u0026ndash;624, Oct. 2016, doi: 10.1016/j.cell.2016.07.055.\u003c/li\u003e\n\u003cli\u003eN. J. White, \u0026ldquo;Malaria parasite clearance,\u0026rdquo; \u003cem\u003eMalar. J.\u003c/em\u003e, vol. 16, no. 1, p. 88, Feb. 2017, doi: 10.1186/s12936-017-1731-1.\u003c/li\u003e\n\u003cli\u003eM. Marti, R. T. Good, M. 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Crystallogr.\u003c/em\u003e, vol. 26, no. 2, Art. no. 2, Apr. 1993, doi: 10.1107/S0021889892009944.\u003c/li\u003e\n\u003cli\u003e\u0026ldquo;Frontiers | Structural Biology in the Clouds: The WeNMR-EOSC Ecosystem.\u0026rdquo; https://www.frontiersin.org/articles/10.3389/fmolb.2021.729513/full (accessed May 09, 2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-proteins-and-proteomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of Proteins and Proteomics](https://www.springer.com/journal/42485)","snPcode":"42485","submissionUrl":"https://submission.nature.com/new-submission/42485/3","title":"Journal of Proteins and Proteomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Malaria, Plasmodium falciparum, CBP2, CX3CL1, ATP","lastPublishedDoi":"10.21203/rs.3.rs-2985578/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2985578/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (Pf) causes the most fatal form of malaria owing to its ability to cytoadhere in the microvasculature of various organs in the body. In addition to the Pf erythrocyte membrane protein 1 (PfEMP1) family that binds diverse host receptors, CX3CL1 binding proteins 1 and 2 (CBP1 and 2) also bind the endothelial chemokine \u0026lsquo;CX3CL1\u0026rsquo; to effect cytoadhesion of parasite infected erthrocytes. CBP2 is a multifaceted protein that binds nucleic acids, Pf skeleton binding protein (PfSBP1) and ATP. ATP binding to the cytoplasmic domain of CBP2 (cCBP2) induces structural changes in the protein, and hints at its role in cell signaling. In this study, we have attempted to identify the ATP binding pocket of CBP2 using an \u003cem\u003ein silico\u003c/em\u003e approach. We have also delineated the type of interactions and amino acid residues that are likely to bind ATP. As CX3CL1 binding proteins are central to parasite biology, the obtained information is likely to form the basis for inhibitor and drug design against this molecule.\u003c/p\u003e","manuscriptTitle":"Investigating the ATP binding pocket of CX3CL1 binding protein 2 using in silico approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-25 16:52:26","doi":"10.21203/rs.3.rs-2985578/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2023-12-26T03:48:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-24T07:59:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"04abd02d-e2b8-43ae-a82f-3f1674e1ff8a","date":"2023-06-14T07:48:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-06-14T04:06:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-29T18:13:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-05-27T00:47:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Proteins and Proteomics","date":"2023-05-26T12:12:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-proteins-and-proteomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of Proteins and Proteomics](https://www.springer.com/journal/42485)","snPcode":"42485","submissionUrl":"https://submission.nature.com/new-submission/42485/3","title":"Journal of Proteins and Proteomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b57ef4fc-dee7-4fb9-9744-cf3d9cdb538e","owner":[],"postedDate":"August 25th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-17T09:28:31+00:00","versionOfRecord":{"articleIdentity":"rs-2985578","link":"https://doi.org/10.1007/s42485-024-00133-z","journal":{"identity":"journal-of-proteins-and-proteomics","isVorOnly":false,"title":"Journal of Proteins and Proteomics"},"publishedOn":"2024-03-26 09:28:31","publishedOnDateReadable":"March 26th, 2024"},"versionCreatedAt":"2023-08-25 16:52:26","video":"","vorDoi":"10.1007/s42485-024-00133-z","vorDoiUrl":"https://doi.org/10.1007/s42485-024-00133-z","workflowStages":[]},"version":"v1","identity":"rs-2985578","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2985578","identity":"rs-2985578","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}