Physicochemical Methods for Protein Function Prediction

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Physicochemical Methods for Protein Function
Prediction

• Mary Jo Ondrechen
• Dept of Chemistry Chemical Biology
• Northeastern University
• Boston, MA 02115

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THEMATICS

• Genomics and proteomics
• About titration curves
• Method for active site location and
  characterization
• Examples
• Future directions and conclusions

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The post-genomic path

• Genome sequence
• Protein sequence
• Protein structure
• Protein function
• Active site location and characterization, drug
  design, understanding protein function, normal
  and disease processes

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PROTEOMICS

• Structural genomics
• rapidly discovering new protein structures, many
  of unknown function
• The Next Frontier
• Characterizing the 106 proteins for which genes
  hold the code.

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Predicting Protein Function

• Protein structure and protein function are not
  well correlated. Need methods to predict
  function from structure (or sequence).
• THEMATICS Theoretical Microscopic Titration
  Curves a reliable way to locate and
  characterize enzyme active sites.

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Typical Experimental Titration Curve
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In the absence of a field, acids obey
Henderson-Hasselbalch

• pH pKa log10A-/HA
• which may be rewritten in terms of the average
  charge as a function of pH
• C _ 10pH / (10pH 10pKa) OR
• C 10pKa / (10pH 10pKa)
• where C is the mean net charge

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C(pH) for a typical residue
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Typical weak acid/base narrow window of
reactivity

• When the pH is close to the pKa, a weak acid/base
  is available to act as both an acid and a base
• By definition of a catalyst, the enzyme must
  regenerate itself before one cycle is over.
• for HA B? ? A? HB,
• reaction proceeds both ways if HA and HB have
  matched pKas

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A common 1st step in enzyme catalysis -

• Deprotonate a C-H bond
• C-H B ? C? HB
• What is required of B?
• It must be a strong enough base
• It must be deprotonated at neutral pH
• Mutually contradictory requirements (for a
  Henderson-Hasselbalch acid/base)

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A better way

• Catalytic base Lysine39 is a very strong base
• AND is partially deprotonated at neutral pH!

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Perturbed titration curves

• Enable residue to act as acid/base over a wide pH
  range
• Precise pH adjustment not needed
• Precise pKa match not required
• Enable residue to have right mix of chemical
  properties
• acid (or base) strength
• right protonation state at neutrality

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Perturbed curves

• Have been noticed before (in titration curves
  obtained computationally for proteins)
• We now understand are significant
• Are markers of chemical reactivity
• Can be used to locate active site
• M.J. Ondrechen, J.G. Clifton and D. Ringe, Proc.
  Natl. Acad Sci USA 98, 12473-12478 (2001)

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THEMATICS

• Theoretical Microscopic Titration Curves
• Conceptually simple
• Require a known structure
• Can be computed
• Highly reliable identifier of active site
• Characterize enzyme active site

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Complementary to other methods

• THEMATICS complements well other methods that
  predict, or provide clues about, function
• Evolutionary history sequence relationships
  sequence homology domain fusion conservation of
  gene position gene coinheritance geometric
  motif search cleft search small molecule
  docking energetics flexibility
• Characterize function by chemical reactivity

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THEMATICS COMPUTATION

• Start with protein structure
• Solve Poisson-Boltzmann equations for electrical
  potential function
• Obtain C(pH) by Monte Carlo method
  Boltzmann-weighted populations
• Plot C(pH) and find perturbed curves

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Which curves are perturbed?

• Visual inspection
• Mathematical analysis (H. Yang)
• Statistical analysis
• Fit to parametrized sigmoid function
• Neural networks / Support Vector Machines (W.
  Tong)
• Only small fraction (3-7) of all ionizable
  residues are perturbed

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Ionizable residues

• Arg Asp Cys Glu His Lys Tyr termini
• A cluster of two or more perturbed residues in
  physical proximity is a reliable predictor of
  active site location
• Success in finding active site is not
  particularly sensitive to selection criteria

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THEMATICS A unique predictive tool for
Proteomics

• Gives chemical information
• Indicates why a particular residue may be
  involved in catalysis
• Highly reliable for identifying active sites
• Conceptually simple
• Computationally (relatively) fast

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Alanine Racemase

• Used by bacteria in cell wall construction
• A target for antibiotics (and for drugs to treat
  tuberculosis)
• Vitamin B6 dependent
• Active as a dimer
• Active site located at dimer interface

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Alanine Racemase catalysis

• Catalyzes interconversion of D-Ala and L-Ala
• Reaction occurs on a Schiff base intermediate
  (alanine pyridoxal phosphate)
• First step on Schiff base remove alpha-H atom
  from Ala moiety
• K39 and Y265 are the catalytic bases

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Alanine Racemase Lysines 39A-234A

• K39 is the catalytic base for D-to-L

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Tyrosines in Alanine racemase
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Results for Alanine Racemase

• Full results for Alanine racemase
• R219, C311, K39, Y43, Y265, Y284, Y354, C358,
  R366, D68
• Bold known active site residue
• Italics second shell
• False positives tend to be isolated

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THEMATICS results

• THEMATICS has succeeded in finding the active
  site for several dozen proteins with a variety of
  folds and chemistries.
• Occasionally, get two or more clusters
• Occasionally, when visual inspection has not
  found a positive cluster, statistical analysis
  has.

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Human Adenosine Kinase

• Catalyzes the transfer of phosphate from ATP to a
  nucleoside analogue
• Unique fold an ?-? three-layer sandwich plus a
  smaller ?-? two-layer domain
• Antiviral and anticancer drug target

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Human Adenosine Kinase

• One of two proteins to date where the human
  observer was unable to locate the active site
• Statistical analysis successful in finding the
  active site (H. Yang)
• Perturbations in predicted titration curves are
  subtle, but statistically significant

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Aspartates in Human AK

• D300 has slightly perturbed curve

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Colicin E3 important test case

• Nuclease - cleaves a phosphodiester linkage in
  the RNA of the ribosome
• Used by e coli to kill rival bacteria
• Unique fold cannot infer active site location
  from other RNAases
• Structure provided by Prof. M. Shoham (CWR) prior
  to publication

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THEMATICS results Colicin E3

• E517, H526, R495, R545, Y519
• Calculation was performed on the structure of the
  catalytic fragment
• Active site found prior to completion of the
  biochemical characterization
• Active site correctly located by THEMATICS

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HIV-1 protease

• Acid protease
• Cathepsin D fold
• Active as a dimer
• D25 and D25 are the catalytic groups
• THEMATICS human observer found D25 and D25

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HIV-1 Protease Aspartates

• Note shape of D25

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THEMATICS on HIV-1 Protease

• Human observer finds
• D25, D25
• Statistical analysis finds
• D25, D25, R8, R87
• R8 and R87 are believed to be involved in
  substrate recognition Bardi, J.S., I. Luque, E.
  Freire, Structure-based thermodynamic analysis of
  HIV-1 protease inhibitors. Biochemistry, 1997.
  36 p. 6588-6596

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Conclusions

• THEMATICS simple, computationally fast, and
  reliable
• Simple connection with chemistry
• A cluster of two or more positive residues is
  predictive of active sites
• Has been automated
• Characterizes residues (reactivity)
• Positive clusters well conserved

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Conclusions - continued

• Perturbed curves result from the polyprotic
  nature of proteins
• Working hypotheses about perturbed titration
  curves
• Afford catalytic advantage
• Afford advantage in reversible binding at
  recognition sites

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Thanks

• David Budil, Leo Murga, Terry Yang, Ying Wei,
  Alissa Bologna, Katie Boino, Wenxu Tong, Bob
  Futrelle, Ron Williams (Northeastern)
• Jaeju Ko (IUP)
• Ihsan Shehadi (UAEU)
• Dagmar Ringe Jim Clifton (Brandeis)

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Support Acknowledged

• National Science Foundation
• Institute for Complex Scientific Software (ICSS)
  - Northeastern

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mjo_at_neu.edu

• M.J. Ondrechen, J.G. Clifton and D. Ringe, Proc.
  Natl. Acad Sci USA 98, 12473-12478 (2001)
• I.A. Shehadi, H. Yang and M.J. Ondrechen, Mol.
  Biol. Rpts 29, 329-335 (2002)