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![30
Using ThreaDom and ProDom, the M protein of (MERS-CoV) had
only one domain (6-199) with Score 912 (355.9 bits), E value 1e-
122 and Identities 165/194 (85%). Moreover, in case of NCBI
conserved domain, there is one domain [ID: cd21567] entitled
Membrane (or Matrix) protein from Middle East respiratory
syndrome-related coronavirus and related beta coronaviruses in
the C lineage with Interval [2-218], E-value [2.46e-156]. This
group contains the Membrane (M) protein of Middle East
respiratory syndrome (MERS)-related CoV, bat-CoV HKU5, and
similar proteins from beta coronaviruses in the merbecovirus
subgenera (C lineage)](https://image.slidesharecdn.com/iman-250314093857-06d737da/75/IMAN-of-medical-microbiology-and-classification-pptx-30-2048.jpg)
![48
The membrane (M) protein is the most abundant structural protein and defines the
shape of the viral envelope, being the central organizer of coronavirus assembly.
The target protein plays a critical role in protein-protein interactions (as well as protein-
RNA interactions) since virus-like particle (VLP) formation in many CoVs requires
only the M and envelope (E) proteins for efficient virion assembly [PMID:25855243].
Interaction of spike (S) with M is necessary for retention of S in the ER-Golgi
intermediate compartment (ERGIC)/Golgi complex and its incorporation into new
virions; binding of M to nucleocapsid (N) proteins stabilizes the nucleocapsid.
Together, M and E proteins make up the viral envelope and their interaction is
sufficient for the production and release of virus-like particles.](https://image.slidesharecdn.com/iman-250314093857-06d737da/75/IMAN-of-medical-microbiology-and-classification-pptx-48-2048.jpg)
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IMAN of medical microbiology and classification.pptx
- 1. 1 Prediction and Analysis ofMiddle East respiratory syndrome- related coronavirus (MERS-CoV) protein by bioinformatics tools Iman Zaghloul Mohamed
- 2.
- 3. 3 Coronaviruses (CoVs)are a large family of enveloped viruses that cause respiratory tract infectious diseases with symptoms similar to the typical common cold .CoVs are categorized into three genus groups, -CoVs, - 𝛼 𝛽 CoVs, and -CoVs. 𝛾 COVID-19 belongs to 𝛽-CoVs similar to SARS and Middle East Respiratory Syndrome (MERS), which are identified as bat-origin that infect people through an intermediate host .
- 4. 4 The MERS-CoV-infectedindividuals develop a cluster of symptoms like fever, shortness of breath, and multi-organ failure in critically ill patients . Since 2012, 27 countries have reported MERS cases, including Austria, Egypt, China, Malaysia, and Bahrain. Between September 2012 and 28 February 2022, a total of 2585 cases, including 891 associated deaths (case fatality rate: 35%) have been globally reported to WHO under the International Health Regulations.
- 5. 5 The most commonCT radiographic finding in hospitalized patients with MERS-CoV infection bilateral predominantly subpleural and basilar airspace changes more extensive ground-glass opacities than consolidation The subpleural and peri broncho vascular predilection of the abnormalities is suggestive of an organizing pneumonia pattern
- 6. 6 Schematic presentation ofthe (MERS-CoV) genome Structure Poly proteins Structural proteins Accessory proteins
- 7. Schematic presentation ofthe (MERS-CoV) genome Structure 7 The average full-length genome sequences of the MERS-CoV based on the (HCoV-EMC/2012-JX869059) is 30 107 bp
- 8. 8 The M proteinof (MERS-CoV) The target protein of this study is RNA-dependent RNA Polymerase (M). M protein plays important role in structure-stabilizing of N protein as it is located in the internal core of virions . It has been demonstrated that M proteins of some CoVs have much higher immunogenicity for T-cell responses than the nonstructural viral proteins .In addition, it plays a critical role in virus-specific B-cell response due to its ability to produce efficient neutralizing antibodies in SARS patients. The M protein of (MERS-CoV) of 219 amino acids (accession number YP_009047210) was retrieved from UniProt Knowledgebase (UniPro t KB), were predicted and analyzed using different bioinformatics prediction tools.
- 9. 9 Using bioinformaticsmethods, we analyzed the MERS CoV E protein’s sequence and its secondary and three- dimensions structures . The goal of protein structure prediction is to estimate the spatial position of every atom of protein molecules from the amino acid sequence by computational methods. Depending on the availability of homologous templates in the PDB library, structure prediction approaches are categorized into template-based modelling (TBM) and free modelling (FM) . Subsequently, the structures of all the sequences in the genome can be modelled using plain comparative modelling methods. In other words, all future protein structure prediction work would be comparative modelling. On the other hand, other structure prediction methods still can be useful in the future; for example, ab initio algorithms can still be used to study the theoretical basis of the protein folding problem .
- 10. Protein structure prediction Importantproteomic tool for understanding phenomena in modern molecular and cell biology and has important applications in biotechnology and medicine. 10 One of these applications is the prediction of three-dimensional structures of virus proteins. This will aid in drug design and integrative understanding of viral processes.
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- 13. 13 Objective 3-D structure predictionof M by using Servers and software according to CASP. Prediction and analysis of motifs and PTMS of M. Functional analysis of M. Structural classification Correlation of Structural and Functional Prediction.
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- 17. 17 1) Multiple sequencealignment. 2) Conserved regions. 3) Phylogenetic analysis. 4) Domain assignment 5) Secondary structures. 6) Three-dimensional structures. 7) Signatures and motifs. 8) Post-translational modification sites. 9) Structural classification. 10) Correlation of Structural and Functional Prediction.
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- 21. 21 1- Multiple sequencealignment
- 22. 22 Multiple sequence alignmentof The M protein of (MERS-CoV) with most homologous proteins using Bio-edit 7.2
- 23. Multiple sequence alignmentof The M protein of (MERS-CoV) with most homologous proteins using Bio-edit 7.2 23
- 24. Multiple sequence alignmentof The M protein of (MERS-CoV) with most homologous proteins using Bio-edit 7.2 24
- 25.
- 26. 26 predicted conserved regionof the M protein of (MERS-CoV) Using bio edit and Clustal omega. Region Position Consensus Segment Length Average entropy (Hx) Region 1 29 to 70 FLLITIVLQYGYPSRSMTVYVFKMFVLWLLWPSSMALSIFSA 42 0.0058 Region 2 95 to 124 SYFVQSIRLFMRTGSWWSFNPETNCLLNVP 30 0.0000 Region 3 130 to 148 VVRPLVEDSTSVTAVVTNG 19 0.0192 Region 4 170 to 213 TVAKPNVLIALKMVKRQSYGTNSGVAIYHRYKAGNYRSPPITAD 44 0.0055
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- 30. 30 Using ThreaDom andProDom, the M protein of (MERS-CoV) had only one domain (6-199) with Score 912 (355.9 bits), E value 1e- 122 and Identities 165/194 (85%). Moreover, in case of NCBI conserved domain, there is one domain [ID: cd21567] entitled Membrane (or Matrix) protein from Middle East respiratory syndrome-related coronavirus and related beta coronaviruses in the C lineage with Interval [2-218], E-value [2.46e-156]. This group contains the Membrane (M) protein of Middle East respiratory syndrome (MERS)-related CoV, bat-CoV HKU5, and similar proteins from beta coronaviruses in the merbecovirus subgenera (C lineage)
- 31.
- 32. Predicted Secondary structureand solvent accessibility of the M protein of (MERS-CoV) using different servers (PredictProtein….Etc.). 32 Secondary 2ry structure Solvent Accessibility Others (Turn/coil/loope) Strand Helix Exposed Buried Percentage (6.39%,35.62% ( 20.09% 37.90% 32% 34%
- 33. 33 6- 3-D structureprediction
- 34. 34 First: Construction ofinitial model using target- template alignment. Second: Reduced-level structure assembly and refinement simulations. Third: Model evaluation and selection.
- 35. Evaluation of 3-Dstructure model from selected servers for NSP12 structure prediction for Domain1 (Cov_RPOL_N). 35 Scores Severs RMSD TM-score QMEAN GDT-HA- score GDT-TS- score Overall Quality Factor Mol- Probity Clash-score Aligned length Ramachandran Favoured 3DREFINE CPH 3.37 0.42034 0.36 0.1244 0.2073 100 2.88 10.0 56 91.46% I-TASSER 1.093 0.9685 0.39 0.1399 00.1762 85.3081 3.83 23.1 184 74.19% PHYER2 3.00 0.94092 0.41 0.0933 0.1347 61.4679 2.85 12.0 118 89.08% SWISS 2.93 0.93103 0.54 0.0881 0.1503 56.044 2.76 17.0 98 91.92% PEP 2.27 0.46683 0.60 0.0052 0.0052 100 3.15 4.8 30 89.47% DEEPREFINE CPH 3.50 0.40373 0.37 0.1244 0.1969 100 3.01 5.7 57 91.89% I-TASSER 0.575 0.9896 0.38 0.1399 0.1762 78.3251 3.38 14.7 185 84.75% PHYER2 3.84 0.92667 0.33 0.0933 0.1347 98.0198 3.16 7.3 118 89.25% SWISS 3.17 0.91307 0.53 0.0881 0.1451 76.6667 3.05 12.0 98 94.52% AlphaFold 3.66 0.70894 -0.24 0.1451 0.2021 94.6602 1.253 1.8 181 95.4% GALAXYREF INE I-TASSER 0.681 0.9858 0.42 0.1399 0.1762 80.1932 2.580 17.80 186 85.71% Galaxy 3.20 0.57742 0.37 0.1244 0.1917 81.9095 1.690 6.89 150 98.16% PHYER2 3.69 0.93423 0.33 0.0933 0.1347 81.8182 2.094 6.39 118 92.44% SWISS 2.97 0.92476 0.54 0.0881 0.1503 77.6471 1.699 9.67 98 98.99% PEP 2.23 0.47415 0.64 0.0052 0.0052 100 2.096 3.28 30 92.11% AlphaFold 3.31 0.69552 -0.31 0.1451 0.2021 93.6893 1.406 7.0 174 99.5% MODREFINE CPH 1.54 0.9848 0.38 0.1244 0.2073 98.6842 2.095 10.0 55 97.56% I-TASSER 1.27 0.9739 0.40 0.1347 0.1710 66.6667 2.92 19.2 185 85.71% Galaxy 1.71 0.9884 0.38 0.1347 0.2073 73.4597 2.45 17.5 151 97.24% PHYER2 3.39 0.9787 0.33 0.0933 0.1347 48.1132 2.55 12.0 118 92.44% SWISS 2.94 0.9931 0.52 0.0881 0.1503 52.6882 2.20 15.7 98 97.98% PEP 0.13618 0.9984 0.59 0.0052 0.0052 100 2.284 8.0 30 97.37% AlphaFold 3.33 0.70216 -0.57 0.1451 0.1969 87.1429 2.106 42.5 176 99.1%
- 36. 36 C B A The best predictedthree-dimensional structure of M protein of (MERS-CoV) by AlphaFold server. (a) Ribbon view of best predicted model, (b) Solvent-accessible surface view showed the exposed regions. (c) The cartoon view showed One block (2-214) in the CoV_M family which was highlighted in green.
- 37.
- 38. 38 The best templatestructure for (Domain1) and (Domain2) of NSP12 of SARS- COV-2 using MUSTER Server and mTM-align server. Scores Domains Subject Tm-Score RMSD Sequence identity Subject length Aligned Length Description Domain1 6nurA 0.7010 0.51 0.602 793 246 SARS-CORONAVIRUS NSP12 BOUND TO NSP7 AND NSP8 CO-FACTORS Domain2 6nurA 0.9974 0.39 0.744 793 442 SARS-CORONAVIRUS NSP12 BOUND TO NSP7 AND NSP8 CO-FACTORS
- 39. 39 7- Signatures andMotifs analysis
- 40. 40 Predicted motifs ofmembrane protein of MERS- CoV using motifs finder, smart and Pfam Server Family POSITION E-value Description Start End Motifs Finder CoV_M 16 211 1.4e-72 Coronavirus M matrix/glycoprotein Smart CoV_M 2 214 4.8e-70 Coronavirus M matrix/glycoprotein Pfam CoV_M 14 214 1.4e-72 Coronavirus M matrix/glycoprotein
- 41.
- 42. 42 Post-translation modification sitesusing HITS-SIB-SWISS motif scan. Category Signature Matching positions RNA Associated Protein Domain Post-Translational Modifications N-glycosylation site. 3-6 cGMP-dependent protein kinase phosphorylation site. 182-185 N-myristoylation site. (81-86,106-111,152-157,187-192) Protein kinase C phosphorylation site. 98-100 Coronavirus M matrix/glycoprotein 1-206 NADH dehydrogenase 24-36 Pseudomurein-binding repeat 160-169 pfam_ls: Corona_M Coronavirus M matrix/glycoprotein 1-219
- 43.
- 44. 44 Scop classification ofM-protein of MERS-CoV 2 Name Sequence Start End Superfamily Family E-value SCOP: d1l9na4 d1l9na4 19 113 Cysteine proteinases Transglutaminase core 3.5 SCOP: d1eysh1 d1eysh1 122 147 PRC-barrel domain Photosynthetic reaction centre, H-chain, cytoplasmic domain family 9.9
- 45. 45 10- Correlation ofStructural and Functional Prediction.
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- 47.
- 48. 48 The membrane (M)protein is the most abundant structural protein and defines the shape of the viral envelope, being the central organizer of coronavirus assembly. The target protein plays a critical role in protein-protein interactions (as well as protein- RNA interactions) since virus-like particle (VLP) formation in many CoVs requires only the M and envelope (E) proteins for efficient virion assembly [PMID:25855243]. Interaction of spike (S) with M is necessary for retention of S in the ER-Golgi intermediate compartment (ERGIC)/Golgi complex and its incorporation into new virions; binding of M to nucleocapsid (N) proteins stabilizes the nucleocapsid. Together, M and E proteins make up the viral envelope and their interaction is sufficient for the production and release of virus-like particles.
- 49.
- 50. Retrieve fasta format MSA, Conserved regions Phylogenetic analysis Domain Separation Secondary structure, Solvent accessibility Tertiary structure prediction Motifs prediction PTMS Structural classification Correlation 4 Conserved regions Family, Superfamily Folds to protein Best model fromI-Tasser and AlphaFold One block (2-214) was detected One Domain 28 Active sites predicted
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