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Olga V. Kalinina Pavel S. Novichkov Andrey A. Mironov Mikhail S. Gelfand

SDPpred : a method for identification of amino acid residues that determine differences in functional specificity of homologous proteins and application thereof to the MIP family of membrane transporters. Olga V. Kalinina Pavel S. Novichkov Andrey A. Mironov Mikhail S. Gelfand

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Olga V. Kalinina Pavel S. Novichkov Andrey A. Mironov Mikhail S. Gelfand

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  1. SDPpred: a method for identification of amino acid residuesthat determine differences in functional specificity of homologous proteins and application thereof to the MIP family of membrane transporters Olga V. Kalinina Pavel S. Novichkov Andrey A. Mironov Mikhail S. Gelfand Aleksandra B. Rakhmaninova

  2. Large families of proteins: generally similar biochemical function but many different specificities… • Example: ~800 transcription factors of the LacI family. • Average sequence identity 30%. • Bind different effectors and operators. • Some effectors: • lactose (LacI) • D-fructose-6-phosphate (FruR) • guanine, hypoxantine (PurR) • cytidine, adenosine (CytR) • trehalose-6-phosphate (TreR) • D-gluconate (GntR) • D-galactose (GalR) • D-ribose (RbsR) • maltose (MalR) • raffinose(RafR) • ……. • Х??

  3. Q9KDW9 ----------MSPFLGEVIGTMILIILGGGVVAGVVLKGTK Q8Y6Z1 ----MIDTSLATQFLGEVIGTAILIILGAGVVAGVSLKRSK Q97JG6 ----------MTIFFAELVGTLLLILLGDGVVANVVLKNSK GLPF_ECOLI MSQT---STLKGQCIAEFLGTGLLIFFGVGCVA--ALKVAG Q8ZJK5 MSQTA-SSTLKGQCIAEFLGTGLLIFFGAGCVA--ALKLAG GLPF_HAEIN MDKS-----LKANCIGEFLGTALLIFFGVGCVA—-ALKVAG GLPF_PSEAE MTTAAPTPSLFGQCLAEFLGTALLIFFGTGCVA--ALKVAG AQPZ_BRUME ---------MLNKLSAEFFGTFWLVFGGCGSAILAA--AFP Q92NM3 ---------MFRKLSVEFLGTFWLVLGGCGSAVLAA--AFP Q8UJW4 ---------MGRKLLAEFFGTFWLVFGGCGSAVFAA--AFP AQPZ_ECOLI ---------MFRKLAAECFGTFWLVFGGCGSAVLAA--GFP Description of specificity groups : Group А: No. 1-10,13… Group В: No.12, 14-16… Group С: No. 17-45… … Alignment SDPpred Testing on families that include proteins with resolved 3D structure Positions that account for specificity Assignment of specificity to new proteins ? Experiment

  4. What are SDPs? (SDP = Specificity Determining Position) • Specificity group = group of proteins that have the same specificity (experimental data, genome analysis, etc.) • SDP = alignment position that is conserved within specificity groups but differs between them SDPis not equivalent to a functionally important position!

  5. Algorithm N - number of groups, - fraction of proteins in groupi. - ratio of occurrences of amino acid In groupiin positionpto the length of the whole alignment column, - frequency of amino acidin the whole alignment column in position p, • Mutual informationIpreflect the extent to which an alignment position tends to be a SDP. • Statistical significance ofIp. Expected mutual information Ipexpof an alignment column. Z-score. (Mirny&Gelfand, 2002, J Mol Biol, 321(1)) • Smoothed amino acid frequencies:a leucine is more a methionine than a valine, and any arginine has a dash of lysine… • Are 5 SDP with Z-score >10.5 better than 10 SDP with Z-score >9.0?Bernoulli estimator for selection of proper number of SDPs • ы …

  6. Kalinina OV, Mironov AA, Gelfand MS, Rakhmaninova AB.(2004) Automated selection of positions determining functional specificity of proteins by comparative analysis of orthologous groups in protein families. Protein Sci 13(2): 443-56 • http://math.belozersky.msu.ru/~psn/ Kalinina OV, Novichkov PS, Mironov AA, Gelfand MS, Rakhmaninova AB. (2004) SDPpred: a tool for prediction of amino acid residues that determine differences in functional specificity of homologous proteins. Nucl Acids Res 32(Web Server issue): W424-8.

  7. Web interface Input: multiple alignment of proteins divided into specificity groups === AQP === %sp|Q9L772|AQPZ_BRUME -------------------------------------mlnklsaeffgtfwlvfggcgsa ilaa--afp-------elgigflgvalafgltvltmayavggisg--ghfnpavslgltv iiilgsts------------------------------slap------------------ qlwlfwvaplvgavigaiiwkgllgrd--------------------------------- ------ %sp|P48838|AQPZ_ECOLI -------------------------------------mfrklaaecfgtfwlvfggcgsa vlaa--gfp-------elgigfagvalafgltvltmafavghisg--ghfnpavtiglwa lvihgatd------------------------------kfap------------------ qlwffwvvpivggiiggliyrtllekrd-------------------------------- ------ %tr|Q92ZW9 -------------------------------------mfkklcaeflgtcwlvlggcgsa vlas--afp-------qvgigllgvsfafgltvltmaytvggisg--ghfnpavslglav iiilgsth------------------------------rrvp------------------ qlwlfwiaplfgaaiagivwksvgeefrpvd----------------------------- ------ === GLP === %sp|P11244|GLPF_ECOLI ----------------------------msqt---stlkgqciaeflgtglliffgvgcv aalkvag---------a-sfgqweisviwglgvamaiyltagvsg--ahlnpavtialwl glilaltd------------------------------dgn--------------g-vpr -flvplfgpivgaivgafayrkligrhlpcdicvveek--etttpseqkasl-------- ------ %sp|P44826|GLPF_HAEIN ----------------------------mdks-----lkancigeflgtalliffgvgcv …

  8. Web interface Output Detailed description of each SDP (List of SDPs) Plot of probabilities, used by the Bernoulli estimator to set the cutoff (Probability plot view) Alignment of the family with the SDPs highlighted (Alignment view)

  9. Examples: the LacI family of bacterial transcription factors • Training set: 459 sequences, average length: 338 amino acids, 85 specificity groups – 44 SDPs 10residues contactNPF (analog of the effector) 7 residues in the effector contact zone (5Ǻ<dmin<10Ǻ) 6 residues make up intersubunit contacts 5 residues in the intersubunit contact zone (5Ǻ<dmin<10Ǻ) 7residues contact the operator sequence 6 residues in the operator contact zone (5Ǻ<dmin<10Ǻ) LacI from E.coli

  10. Examples: bacterial membrane channels of the MIP family • Training set: 17 sequences, average length 280 amino acids, 2 specificity groups: Aquaporines & glyceroaquaporines – 21 SDPs 8 residues contact glycerol (substrate) (dmin<5Ǻ) 8residues oriented to the channel GlpF from E.coli 5 residues make up contacts with other subunits

  11. Total 348 amino acids 44 SDP Why does the prediction make sense?LacI fromE.coli Non-contacting residues (distance to the DNA, effector, or the other subunit >10Ǻ) Contact zone (may be functional) Contacting residues (distance to the DNA, effector, or the other subunit <5Ǻ)

  12. Total 281 amino acids 21 SDP Why does the prediction make sense?GlpF fromE.coli Non-contacting residues (distance to the substrate, or another subunit >10Ǻ) Contact zone (may be functional) Contacting residues (distance to the substrate, or another subunit <5Ǻ)

  13. GlpF fromE.coli,a membrane channel from the MIP family: SDPs either interact with the substrate or are located on the outer surface of the monomer Structure of theGlpF monomer Predicted SDPs Glycerol

  14. SDPs located on the outer surface of the GlpF monomer form subunit contacts 20Leu, 24Ile, 108Tyrof one subunit, 193Serfrom another subunit Glu43 from all four subunits

  15. SDPs located on the outer surface of the GlpF monomer(continued)

  16. SDPs located on the outer surface of the GlpF monomer(continued) Structure of contacts in the type B cluster Structure of contacts in the type A cluster

  17. ConclusionsI.SDPpred: the SDP prediction method • A method for identification of amino acid residues that account for differences in protein functional specificity • Does not rely on the protein 3D structure • Automatically determines the number of significant positions • Considers substitutions according to the chemical properties of substituted amino acids • Results agree with available structural and experimental data • Applicable to any protein family in a standard way Kalinina OV, Mironov AA, Gelfand MS, Rakhmaninova AB.(2004) Automated selection of positions determining functional specificity of proteins by comparative analysis of orthologous groups in protein families. Protein Sci 13(2): 443-56 http://math.belozersky.msu.ru/~psn/ Kalinina OV, Novichkov PS, Mironov AA, Gelfand MS, Rakhmaninova AB. (2004) SDPpred: a tool for prediction of amino acid residues that determine differences in functional specificity of homologous proteins. Nucl Acids Res 32(Web Server issue): W424-8.

  18. ConclusionsII.SDPsforGlpF fromE.coli • In protein families, whose members function as oligomers,predicted SDPs are often localized on the contact surface between subunits • 5 “surface” SDPs inGlpF: 20Leu, 24Ile, 43Glu, 108Tyr, 193Ser. All of them participate in forming the quaternary structure • Evolutionary pressure on amino acids that establish intersubunit contacts correlates with evolutionary pressure on amino acids that account for the correct recognition of the substrate • These residues form compact spatial clusters • “structural clasps” for recognition of proper subunits

  19. Olga V. Kalinina Pavel S. Novichkov Andrey A. Mironov Mikhail S. Gelfand Aleksandra B. Rakhmaninova Department of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia Institute for Information Transmission Problems RAS, Moscow, Russia State Scientific Center GosNIIGenetika, Moscow, Russia Acknowledgements Leonid A. Mirny Olga Laikova Vsevolod Makeev Roman Sutormin Shamil Sunyaev Aleksey Finkelstein

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