Intersubunit contacts are often facilitated by specificity-determining positions

1
Intersubunit contacts are often facilitated by
specificity-determining positions

• Computational identification of protein positions
  that possibly account for precise recognition of
  the interaction partner

2

• Abundance of sequence data
• Little experimental information on protein
  function
• gt annotation by homology
• Even less information on protein specificity
• gt prediction of specificity-determining
  positions (SDPs)

3
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!
4
What can we infer from SDPs?

• Targets for protein functional redesign
• Specificity signature
• Sites of protein-protein interaction

5
Talk overview

• SDPpred, an algorithm for identification of SDPs
• A studied example isocitrate/isopropylmalate
  dehydrogenases
• Link to PPI

6
SDPpred

• Multiple protein alignment divided into
  specificity groups

AQP spQ9L772AQPZ_BRUME ----------------
---------------------mlnklsaeffgtfwlvfggcgsa ilaa-
-afp-------elgigflgvalafgltvltmayavggisg--ghfnpavs
lgltv iiilgsts------------------------------slap--
---------------- qlwlfwvaplvgavigaiiwkgllgrd------
--------------------------- ------ GLP
spP11244GLPF_ECOLI ------------------------
----msqt---stlkgqciaeflgtglliffgvgcv aalkvag------
---a-sfgqweisviwglgvamaiyltagvsg--ahlnpavtialwl gl
ilaltd------------------------------dgn-----------
---g-vpr -flvplfgpivgaivgafayrkligrhlpcdicvveek--e
tttpseqkasl-------- ------
SDPpred
SDPs positions best discriminating between
specificity groups
7
What is in the black box the algorithm

• Mutual information Ip reflect the extent to which
  an alignment position tends to be a SDP.
• Statistical significance of Ip.
• Expected mutual information Ipexp of an
  alignment column.
• Z-score.
• (MirnyGelfand, 2002, J Mol Biol, 321(1))
• Are 5 SDP with Z-score gt10.5 better than 10 SDP
  with Z-score gt9.0? Bernoulli estimator for
  selection of proper number of SDPs
• Smoothed amino acid frequencies a leucine is
  more a methionine than a valine, and any arginine
  has a dash of lysine

- ratio of occurences of amino acid a in group i
in position p to the height of the alignment
column - frequency of amino acid a in position
p - fraction of proteins in group i
8
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)

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Special features of SDPpred

• Smoothed amino acid frequencies allow to account
  for functional (structural, chemical,
  evolutionary, ) similarities among amino acids
• Automatic cutoff setting -gt no prior knowledge
  about protein family
• Does not require 3D structure -gt 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/

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Example isocitrate/isopropylmalate
dehydrogenases (IDH/IMDH)

• IDH catalyzes the oxidation of isocitrate to
  a-ketoglutorate and CO2 (TCA) using either NAD or
  NADP as a cofactor in different organisms from
  bacteria to higher eukaryotes
• IMDH catalyzes oxidative decarboxylation of
  3-isopropylmalate into 2-oxo-4-methylvalerate
  (leucine biosynthesis) in bacteria and fungi

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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
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IDH/IMDH selecting specificity groups

1. All NAD-dependent vs. all NADP-dependent

1. All IDHs vs. all IMDHs

1. Four groups

IDH (NADP) type II
IDH (NADP) type II
IDH (NADP) type II
IDH (NAD)
IDH (NAD)
IDH (NAD)
IMDH (NAD)
IMDH (NAD)
IMDH (NAD)
IDH (NADP) type I
IDH (NADP) type I
IDH (NADP) type I
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IDH/IMDH predicted SDPs (cofactor-specific)
Substrate
Cofactor
SDPs
Subunit I
Subunit II
NADP-dependent IDH from E. coli (1ai2)
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IDH/IMDH predicted SDPs (substrate-specific)
Substrate
Cofactor
SDPs
Subunit I
Subunit II
NADP-dependent IDH from E. coli (1ai2)
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IDH/IMDH predicted SDPs (four groups)
Substrate
Cofactor
SDPs
Subunit I
Subunit II
NADP-dependent IDH from E. coli (1ai2)
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IDH/IMDH predicted SDPs (overview)
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IDH/IMDH SDPs predicted for different groupings
All NAD-dependent vs. all NADP-dependent -gt
cofactor-specific SDPs
All IDHs vs. all IMDHs -gt substrate-specific SDPs
208Arg
337Ala
100Lys
300Ala
105Thr
341Thr
229His
154Glu
103Leu
233Ile
97Val
158Asp
115Asn
305Asn
308Tyr
98Ala
155Asn
231Gly
327Asn
287Gln
344Lys
164Glu
345Tyr
351Val
241Phe
38Gly
40Asp
104Thr
Color code Contacts substrate Contacts
cofactor Contacts the other subunit Contacts
substrate AND cofactor Contacts substrate AND the
other subunit
107Val
152Phe
161Ala
232Asn
245Gly
323Ala
31Tyr
36Gly
162Gly
Four groups
45Met
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IDH/IMDH SDPs in contact with cofactor
Substrate (isocitrate)
100Lys, 104Thr, 105Thr, 107Val, 337Ala,
341Thr substrate-specific and four group SDPs,
functionally not characterized
Cofactor (NADP)
Nicotinamide nucleotide
Adenine nucleotide
344Lys, 345Tyr, 351Val cofactor-specific
SDPs, known determinants of specificity to
cofactor
NADP-dependent IDH from E. coli (1ai2)
19
Clusters of SDPs on the intersubunit contact
surface

• in the IDH/IMDH family

Cluster II
Cluster I
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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)
21

• Bacterial membrane transporters from the MIP
  family
• Water and glycerol/water channels

Cluster II
Cluster I
Substrate (glycerol)
Subunit I
Glpf (glycerol facilitator) from E. coli (1fx8)
22
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

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• 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!