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Intersubunit contacts are often facilitated by specificity-determining positions Computational identification of protein positions that possibly account for precise recognition of the interaction partner Abundance of sequence data Little experimental information on protein function => annotation by homology Even less information on protein specificity => prediction of specificity-determining positions (SDPs) SDP (Specificity-Determining Position) Alignment position that is conserved within groups of proteins having the same specificity (specificity groups) but differs between them SDP is not equivalent to a functionally important position! What can we infer from SDPs? Targets for protein functional redesign Specificity signature Sites of protein-protein interaction Talk overview SDPpred, an algorithm for identification of SDPs A studied example: isocitrate/isopropylmalate dehydrogenases Link to PPI SDPpred Multiple protein alignment divided into specificity groups === AQP === %sp|Q9L772|AQPZ_BRUME -------------------------------------mlnklsaeffgtfwlvfggcgsa ilaa--afp-------elgigflgvalafgltvltmayavggisg--ghfnpavslgltv iiilgsts------------------------------slap-----------------qlwlfwvaplvgavigaiiwkgllgrd-------------------------------------… === GLP === %sp|P11244|GLPF_ECOLI ----------------------------msqt---stlkgqciaeflgtglliffgvgcv aalkvag---------a-sfgqweisviwglgvamaiyltagvsg--ahlnpavtialwl glilaltd------------------------------dgn--------------g-vpr -flvplfgpivgaivgafayrkligrhlpcdicvveek--etttpseqkasl------------… SDPpred SDPs: positions best discriminating between specificity groups What is in the black box: the algorithm Mutual information Ip reflect the extent to which an alignment position tends to be a SDP. Ip f p ( , i) log all specificity all amino groupsi acids f p ( , i) f p ( ) f (i) f p ( , i ) - ratio of occurences of amino acid α in group i in position p to the height of the alignment column - frequency of amino acid α in position p - fraction of proteins in group i f p ( ) f (i ) Statistical significance of Ip. Expected mutual information Ipexp of an alignment column. Z-score. I p I exp p Z p (I exp) p (Mirny&Gelfand, 2002, J Mol Biol, 321(1)) 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 Z1 Z 2 k * arg min Pthere are at least k observed Z - scores Z Z k k n arg min 1 C ni q i p n i k i n k 1 p P( Z Z k ) Zk 1 exp( Z 2 )dZ 2 q 1 p Smoothed amino acid frequencies: a leucine is more a methionine than a valine, and any arginine has a dash of lysine… f ( , i) n( , i) n(i) 20 n( , i) n( , i)m( ) ~ 1 f ( , i) n(i) n(i) n(i) Other similar techniques Evolutionary trace (Lichtarge et al. 1996, 1997) Evolutionary rate shifts (Gaucher et al. 2002) Surface patches of slowly evolving residues (Rate4Site, Pupko et al. 2002) PCA in sequence space (Casari et al. 1999, del Sol Mesa et al. 2003) Correlated mutations (Pazos and Valencia, 2002) Prediction of functional sub-types (Hannenhalli and Russell, 2000) and identification of PSDR (Mirny and Gelfand, 2002) Special features of SDPpred Smoothed amino acid frequencies allow to account for functional (structural, chemical, evolutionary, …) similarities among amino acids Automatic cutoff setting -> no prior knowledge about protein family Does not require 3D structure -> use of structural data solely for interpretation and verification of results – Kalinina OV, Mironov AA, Gelfand MS, Rakhmaninova AB. (2004) Protein Sci 13(2): 443-56 – Kalinina OV, Novichkov PS, Mironov AA, Gelfand MS, Rakhmaninova AB. (2004) Nucl Acids Res 32(Web Server issue): W424-8. – http://math.belozersky.msu.ru/~psn/ Example: isocitrate/isopropylmalate dehydrogenases (IDH/IMDH) IDH: catalyzes the oxidation of isocitrate to αketoglutorate and CO2 (TCA) using either NAD or NADP as a cofactor in different organisms from bacteria to higher eukaryotes IMDH: catalyzes oxidative decarboxylation of 3isopropylmalate into 2-oxo-4-methylvalerate (leucine biosynthesis) in bacteria and fungi IDH/IMDH: combinations of specificities towards substrate and cofactor NAD-dependent IDHs NADP-dependent IDHs from bacteria and archaea (type I) NADP-dependent IDHs from eukaryota (type II) NAD-dependent IMDH Eukaryota Archaea Bacteria Eukaryota Mitochondria Archaea Bacteria IDH/IMDH: selecting specificity groups 1. All NAD-dependent 2. All IDHs vs. all vs. all NADPdependent IMDHs IDH (NADP) type II IDH (NAD) IDH (NADP) type I Four groups IDH (NADP) type II IDH (NAD) IMDH (NAD) 3. IDH (NADP) type II IDH (NAD) IMDH (NAD) IDH (NADP) type I IMDH (NAD) IDH (NADP) type I IDH/IMDH: predicted SDPs (cofactor-specific) Substrate Cofactor SDPs Subunit I Subunit II NADP-dependent IDH from E. coli (1ai2) IDH/IMDH: predicted SDPs (substrate-specific) Substrate Cofactor SDPs Subunit I Subunit II NADP-dependent IDH from E. coli (1ai2) IDH/IMDH: predicted SDPs (four groups) Substrate Cofactor SDPs Subunit I Subunit II NADP-dependent IDH from E. coli (1ai2) IDH/IMDH: predicted SDPs (overview) IDH/IMDH: SDPs predicted for different groupings All NADdependent vs. all NADP-dependent -> cofactorspecific SDPs 208Arg 337Ala 100Lys 300Ala Color code: 105Thr 229His 154Glu 103Leu 233Ile 158Asp 115Asn 305Asn 308Tyr 155Asn 231Gly 327Asn 344Lys 287Gln 164Glu 351Val 345Tyr 241Phe 38Gly 40Asp 104Thr 107Val 152Phe 323Ala 245Gly 161Ala 232Asn Contacts substrate Contacts cofactor 162Gly 36Gly Contacts the other subunit 45Met Contacts substrate AND cofactor Contacts substrate AND the other subunit All IDHs vs. all IMDHs -> substratespecific SDPs 31Tyr 341Thr 97Val 98Ala Four groups IDH/IMDH: SDPs in contact with cofactor Substrate (isocitrate) Cofactor (NADP) Nicotinamide nucleotide 100Lys, 104Thr, 105Thr, 107Val, 337Ala, 341Thr: substrate-specific and four group SDPs, functionally not characterized Adenine nucleotide 344Lys, 345Tyr, 351Val: cofactor-specific SDPs, known determinants of specificity to cofactor NADP-dependent IDH from E. coli (1ai2) Clusters of SDPs on the intersubunit contact surface in the IDH/IMDH family… Cluster II Cluster I …and in other protein families The LacI family of bacterial transcription factors Bind specific operator sequences upon interaction with effector molecules, mainly various sugars Cluster I Effector Cluster II DNA operator LacI (lactose repressor) from E.coli (1jwl) Bacterial membrane transporters from the MIP family Water and glycerol/water channels Cluster I Cluster II Substrate (glycerol) Subunit I Glpf (glycerol facilitator) from E. coli (1fx8) Conclusions SDPpred, a method for identification of amino acids that account for differences in protein specificity Results obtained for several protein families of different functional type agree with structural and experimental data A substantial fraction of SDPs are located on the intersubunit contacts interface, where they form distinct spatial clasps 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 Thank you!