Tyrosine-protein+phosphatase+CpsB+(Streptococcus+pneumoniae)


 * *Target (protein/gene name): ** Tyrosine-protein phosphatase CpsB


 * *NCBI Gene # or RefSeq#: ** [|WP_000565348.1.]


 * *Protein ID (NP or XP #) or Wolbachia#: ** (No NP, XP, or Wolbachia # found): [|WP_000565348.1.]


 * *Organism (including strain): ** Streptococcus pneumoniae, strain 19f, TIGR4


 * Etiologic Risk Group (see link below): ** RG2

Streptococcus pneumoniae is a Gram-positive member of the genus Streptococcus. This bacterium is a lancet-shaped, gram-positive bacteria, which can grow in pairs or in short chains. The main pathogenicity of the pneumococci is due to the many different structures found on the surface of the bacterial cell. When the pneumococcus cells come into contact with mucus secretions, the capsule is used to help reduce the entrapment within the mucus, and allowing the bacterium to enter the epithelial surfaces in the upper-respiratory tract. This ability can be attributed to the capsule’s negatively charged polysaccharides, allowing repulsion from the sialic acid-rich mucopolysaccharides that are in mucus. Furthermore, the capsule allows the bacterial cell to be resistant to phagocytosis by phagocytes in the human body, allowing for greater infection potential. Once on the epithelial cells of the alveolar sacs within the lungs, the cell wall of the bacteria cause inflammation because of the purified peptidoglycan that makes up the cell wall. The cell wall also activates the alternative complement pathway, promoting the activity of C3 proteins and causing anaphylatoxins production. This further activates the body’s immune response causing greater inflammation and symptoms to develop.
 * */ Disease Information : **

After multiplication of the bacteria in the lungs, pneumococcal lysis will occur through the activation of the enzyme autolysin, which is present in the bacterium, releasing cell wall products and pneumolysin into the surrounding area. Pneumolysin is then responsible for cell lysis at high concentrations and as a toxin at any other concentration. In its toxin form, this protein can inhibit the epithelial cells’ ciliary movement, bactericidal activity, and increased white blood cell count. From that point, the bacteria will continue to multiply and lyse. All of this activity between the bacteria and the immune system causes the definitive pleural effusions that can be seen on x-ray images, and the cell lysis causes further inflammatory response as more anaphylatoxins are produced. Some notable symptoms caused by these responses are fever, sweating, shaking chills, coughing that can produce phlegm, chest pain when breathing or coughing, and shortness of breath. Therefore, the constant cycle of multiplication, lysis, and inflammatory responses increase the effects of infection exponentially.

Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):: Monomer as a biological unit. fascioquinol E @http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356977/ [] Fig. 1: BRENDA reaction mechanism of tyrosine-protein phosphatase (E.C.: 3.1.3.48)
 * Link to TDR Targets page (if present): ** None
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) PATRIC NCBI **
 * Essentiality of this protein: ** This protein is essential as it is involved in the capsule polysaccharide biosynthesis pathway. Through the catalytic activity of this protein, the organism is able to perform systemic virulence on its host, dephosphorylate CpsD to proceed with capsule formation, and therefore invade, attack, and multiply.
 * Complex of proteins?: ** No, monomer
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **
 * *EC#: ** 3.1.3.48
 * Link to BRENDA EC# page: **

@https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/ptp101bul.pdf @http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097125/ @http://www.sigmaaldrich.com/catalog/product/sigma/ptp101?lang=en&region=US Linked kit has been discontinued. Individual reagents' prices not listed.
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **
 * -- -or link (or citation) to paper that contains assay information **
 * -- links to assay reagents (substrates) pages. **
 * --- List cost and quantity of substrate reagents, supplier, and catalog # **

-- PDB # or closest PDB entry if using homology model: 2WJD_A Fig. 2: NCBI BLASTP result of tyrosine-protein phosphatase (UniProtKB:Q54518) against PDB with top match identified as 2WJD Chain A.
 * Structure (PDB or Homology model) **

Max % Identities: 87% % Positives: 93% Chain used for homology: Chain A

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777267/#bib41
 * Current Inhibitors: ** Samarium (III) ion, Sulfate ion, Manganese (II) ion
 * Expression Information (has it been expressed in bacterial cells): ** Purified and extracted from E. coli Rosetta cells. CpsB gene was amplified by PCR and cloned into the pEHISTEV vector.
 * Purification Method : ** His-tag Ni-NTA affinity purification


 * Image of protein (PyMol with features delineated and shown separately): **

Fig. 3: PyMol representation of 2WJD Chain A shown as sticks and colored by element with carbon green. Surface transparency set to 80%.

MIDIHSHIVFDVDDGPKSREESKALLAESYRQGVRTIVSTSHRRKGMFETPEEKIAENFL
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **

QVREIAKEVADDLVIAYGAEIYYTLDALEKLEKKEIPTLNDSRYALIEFSMHTSYRQIHT

GLSNILMLGITPVIAHIERYDALENNEKRVRELIDMGCYTQINSYHVSKPKFFGEKYKFM

KKRARYFLERDLVHVVASDMHNLDSRPPYMQQAYDIIAKKYGAKKAKELFVDNPRKIIMD

QLI


 * *length of your protein in Amino Acids: ** 247 aa
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website: ** 28447.6 g/mol
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 17880 L mol-1 cm-1

Fig. 4: TMpred output fo amino acid sequence of protein 2WJD chain A. Graph shows a representation of TMpred program's prediction of membrane-spanning regions and their orientation.
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

1 midihshivf dvddgpksre eskallaesy rqgvrtivst shrrkgmfet peekiaenfl
 * *CDS Gene Sequence (paste as text only): **

61 qvreiakeva ddlviaygae iyytldalek lekkeiptln dsryaliefs mhtsyrqiht

121 glsnilmlgi tpviahiery dalennekrv relidmgcyt qinsyhvskp kffgekykfm

181 kkraryfler dlvhvvasdm hnldsrppym qqaydiiakk ygakkakelf vdnprkiimd

241 qli


 * *GC% Content for gene: ** 46.776406%


 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **

1 ATGATCGATATCCACTCTCACATCGTTTTCGACGTTGACGACGGTCCGAAATCTCGTGAA 61 GAGTCTAAAGCGCTGCTGGCGGAATCTTATCGTCAGGGTGTTCGTACCATCGTTAGCACC 121 TCTCACCGCCGCAAAGGTATGTTCGAAACCCCGGAAGAGAAGATCGCGGAAAACTTCCTC 181 CAGGTTCGTGAAATCGCGAAAGAAGTTGCGGACGACCTCGTTATCGCGTATGGCGCTGAA 241 ATCTACTATACCCTGGATGCGCTGGAAAAACTGGAGAAAAAAGAAATCCCGACCCTGAAC 301 GACTCTCGTTACGCGCTCATCGAATTCTCTATGCACACCTCTTACCGTCAGATCCACACC 361 GGTCTGTCTAACATCCTGATGCTGGGTATCACCCCGGTAATCGCCCACATCGAACGTTAC 421 GACGCACTCGAGAACAACGAAAAACGCGTCCGCGAACTGATCGACATGGGCTGCTACACC 481 CAGATCAACTCCTACCACGTTTCTAAACCGAAATTCTTCGGTGAAAAGTACAAGTTTATG 541 AAAAAGCGTGCGCGTTACTTCCTGGAACGTGACCTGGTTCACGTTGTTGCGTCTGACATG 601 CACAATCTGGACTCCCGTCCGCCGTATATGCAGCAGGCGTACGACATCATTGCGAAGAAA 661 TACGGTGCAAAAAAAGCCAAAGAACTCTTCGTTGATAACCCGCGTAAGATCATCATGGAC 721 CAGCTGATCTAA


 * *GC% Content for gene (codon optimized): ** 49.863388%

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): **
 * ( link to DNA Works output text file - ** that should be saved in your Google Docs folder after you did the primer design protocol)

[|DNAWorks Output - CpsB]


 * Primer design results for 'tail' primers (this is just 2 sequences): **

[|TailPrimerDesignProtocoljwk955fall2016]

__ Forward Primer: __

5’ TACTTCCAATCC ATGATCGATATCCACTCTCAC 3’ _ 33 bp

GC Content _ 42.4 _%

__ 0 mM Mg 2+ T m _ 60.6 __ o C1.5 mM Mg 2+ T m _ 68 __ o C 2 mM Mg 2+ T m _ 68.5 __ o C

4 mM Mg 2+ T m _ 69.5 __ o C 6 mM Mg 2+ T m _ 69.9 __ o C

__Reverse Primer__:

5’ ATGGACCAGCTGATCTAA CAGTAAAGGTGGATA 3’

__ Reverse complement it: __

5’ TATCCACCTTTACTG TTAGATCAGCTGGTCCAT 3’ _ 33 __bp__

GC Content _ 42.4 %

0 mM Mg 2+ T m _ 61.8 __ o C1.5 mM Mg 2+ T m _ 69.2 __ o C 2 mM Mg 2+ T m _ 69.7 __ o C __

__ 4 mM Mg 2+ T m _ 70.7 __ o C 6 mM Mg 2+ T m _ 71.2 __ o C

Are your Temps (Tm) within a few degrees? YES

TMPrediction:

= TMpred output for unknown = Sequence: MID...QLI, length: 243 Prediction parameters: TM-helix length between 17 and 33
 * [EMBnet-Server] ** Date: Thu Oct 27 2:27:34 2016

1.) Possible transmembrane helices
The sequence positions in brackets denominate the core region. Only scores above 500 are considered significant. code Inside to outside helices :  1 found from       to    score center 117 ( 117) 135 ( 135)   983    127

Outside to inside helices :  1 found from       to    score center 118 ( 118) 135 ( 135)  1128    127 code

2.) Table of correspondences
code Here is shown, which of the inside->outside helices correspond to which of the outside->inside helices. code > Helices shown in brackets are considered insignificant > A "+"-symbol indicates a preference of this orientation. > A "++"-symbol indicates a strong preference of this orientation. code inside->outside | outside->inside 117- 135 (19) 983      |   118- 135 (18) 1128 code

3.) Suggested models for transmembrane topology
code These suggestions are purely speculative and should be used with ** extreme caution ** since they are based on the assumption that all transmembrane helices have been found. In most cases, the Correspondence Table shown above or the prediction plot that is also created should be used for the topology assignment of unknown proteins. code code 2 possible models considered, only significant TM-segments used

-> STRONGLY prefered model: N-terminus outside 1 strong transmembrane helices, total score : 1128 1 118  135 (18)    1128 o-i
 * 1) from   to length score orientation

--> alternative model 1 strong transmembrane helices, total score : 983 1 117  135 (19)     983 i-o code
 * 1) from   to length score orientation