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Investigating allosteric communication in protein systems with new conformational fluctuation covari

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Michael Bradley, Rachel Rice, Peter Chivers, Enrico Di Cera Nathan Baker ... Rachel Rice. Molecular biophysics grad student. Peter Jones. Programmer. Sun Joo Lee ... – PowerPoint PPT presentation

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Title: Investigating allosteric communication in protein systems with new conformational fluctuation covari


1
Investigating allosteric communication in protein
systems with new conformational fluctuation
covariance analysis methods
  • Michael Bradley, Rachel Rice, Peter Chivers,
    Enrico Di Cera Nathan Baker
  • Dept. of Biochemistry and Molecular Biophysics
  • Molecular Biophysics Graduate Program
  • Center for Computational Biology
  • Washington University in St. Louis
  • http//agave.wustl.edu

2
Baker group
3
Understanding allostery
Predictive mutagenesis and experimental
testing
?
  • Develop a predictive model of
  • Large scale conformational changes
  • Residue/atom-scale conformational changes
  • Communication pathway(s)
  • Ligand specificity and dependency
  • System-dependent?
  • NikR
  • Thrombin

4
Existing methods (a sparse review)
  • Harmonic and quasi-harmonic methods
  • Brooks CL III, et al.
  • McCammon JA, et al.
  • Bahar I, et al. Jernigan RL, et al.
  • Graph- and contact-related methods
  • del Sol A, ..., Nussinov R. Mol Sys Biol (2006)
  • Chennubhotla C, Bahar I. PLoS Comput Biol (2007)
  • Daily MD, Upadhyaya TJ, Gray JJ. Proteins (2007)
  • Ghosh A, Vishveshwara S. PNAS (2007)
  • Energy flow in molecules
  • Sharp K, Skinner JJ. Proteins (2006)
  • Yu X, Leitner DM. J Chem Phys (2005)
  • And many more...

5
Do we need a new method?
  • Problem-specific
  • General approach
  • Goals
  • Want to combine large-scale analyses (PCA, ENM,
    etc.) with microscopic view of sidechains
  • Move correlation matrix to contact level
  • Understand enough about sidechain behavior to
    engineer mutations
  • Eventually... understand energy flow in
    allosteric molecules

6
Methodology overview
  • A myopic view of allostery
  • Sidechain rearrangement
  • Salt-bridge/hydrogen bond formation and
    dissolution
  • Complements large-scale PCA and normal-mode
    methods
  • Designed to provide specific residue-level
    information for mutagenesis
  • Originally conceived as PCA on contact space

7
Contact definitions
  • Two types of contacts
  • Polar
  • Salt bridges
  • Hydrogen bonding
  • Nonpolar
  • Not currently considered
  • Two structures for contacts
  • Sidechains
  • Backbones

Sidechain-sidechain
Backbone-sidechain
Backbone-backbone
8
Contact analyses means
  • Classic analysis technique
  • Probability of contact formation
  • Provides graph of residue-residue contacts
  • Various graph theoretic analyses
  • Connectivity
  • Number of components
  • Strength through second eigenvalue of Laplacian
  • Routing and pathways
  • Site-site fluxes
  • Minimum-weight paths

9
Contact analyses correlations
  • Examines the correlated making and breaking
    of contacts
  • Quantified by binary correlation coefficient
  • Also gives a graph-like structure

10
Correlations and statistical significance
  • Binary correlation coefficient has natural
    connection to ?2 metric (with 1 DOF)
  • Set significance at 95 confidence
  • Set number of observations to times a bond is
    made and broken
  • Result thresholds contact correlation graph

11
Contact correlation analysis hubs
  • Highly connected contacts
  • Correlate with many other contacts in the
    structure
  • Often found in secondary structure elements
  • Functionally important?

12
Contact correlation analysis paths and clusters
  • Paths
  • What is the shortest route between two sites in
    the molecule?
  • Use weighted Dijkstra algorithm
  • Set weights by ?2 or contact occupancies
  • Clusters
  • What is the smallest set of residues which
    correlate with functional site(s)?
  • Use UPGMA clustering method
  • Set weighted similarities by ?2 or contact
    occupancies

13
General applications to allostery
  • Analysis of apo and holo forms
  • Explicitly study conformational changes between
    apo and holo
  • Use contact occupancy graph as tool to examine
    altered connectivity
  • Provides more local view than general metrics of
    conformational change
  • Analysis of apo form only
  • Assume fluctuation-dissipation or population
    shift mechanism
  • Examine apo form for motions which promote
    allosteric change
  • Use contact correlation to probe communication
    pathways
  • Provides more local view than PCA

14
Specific biomolecular applications
  • Method driven by two problems
  • Allosteric activation of NikR by Ni (Chivers)
  • Allosteric activation of thrombin by Na (Di
    Cera)
  • Experimental tests through collaboration

15
Application to NikR allostery
NikR tetramer
Hydrogenases
Ni2
Ni2
Ni2
- Ni2
nik genes
NikR DNA recognition sites
Inner and outer membranes
Rowe JL, Starnes GL, Chivers PT. J Bacteriol 187
(18) 6317-23, 2005.
16
The NikR homotetramer
Schreiter ER, Sintchak MD, Guo Y, Chivers PT,
Sauer RT, Drennan CL. Nat Struct Biol, 10
794-9, 2003 Schreiter ER, Wang SC, Zamble DB,
Drennan CL. PNAS, 103 13676-81, 2006.
17
Questions for NikR function
  • Metal Binding
  • Required for DNA binding (40 Å site separation)
  • Increases NikR stability
  • Causes an average conformational change in
    solution
  • H/D exchange mass spectrometry
  • How do the RHH and ACT domains communicate?
  • Is the mechanism of ACT domain control a general
    feature of the fold?

18
Overview of NikR study
  • Hypothesis the NikR structure encodes
    interdomain communication and is poised to relay
    the Ni binding signal through a set of
    specific interactions. This is a
    fluctuation-dissipation or population shift
    model
  • Approach use simulations of the apo protein to
    examine correlated fluctuations in NikR structure
  • Goal
  • Identify mechanism of NikR allostery
  • Suggest mutations for experimental study

19
Molecular dynamics of apo NikR
  • System
  • Full NikR tetramer- 532 amino acids
  • Periodic box of 28,797 H2O
  • TIP3P water model
  • 150 mM NaCl
  • NpT ensemble 298K, 1.0 atm
  • Amber 8.0 with the Amber99 forcefield
  • 80 ns with 30 ns equilibration

Bradley MJ, Chivers PT, Baker NA. J Mol Biol, in
press.
20
A global view of conformation space
  • PCA modes from simulation show good overlap with
    crystal structure deformations
  • Also agrees with ANM
  • High cooperativity obscures residue-specific
    involvement

apo-holo crystal structure displacement
Molecular dynamics PCA mode 2
Bradley MJ, Chivers PT, Baker NA. J Mol Biol, in
press.
21
A global view of conformation space
  • Trajectory-averaged correlation matrix
  • Shows relative domain motion
  • Identifies highly mobile regions of backbone

22
Maximal weight contact paths
  • High occupancy paths between Ni site and DNA
    binding residues
  • Reflects structural connectivity between sites
  • Includes residues of known functional importance
    as well as new hits
  • No communication in this analysis

23
Contact correlation clusters
  • Highly correlated residues connecting Ni and
    DNA binding sites
  • 3 main clusters involved in communication
  • Includes residues of known functional importance
    as well as new hits

Bradley MJ, Chivers PT, Baker NA. J Mol Biol, in
press.
24
Identifying residues important in allostery
  • Sources of information
  • Correlation hubs and clusters
  • Pathways
  • Verification of hits
  • Sequence conservation
  • Experimental data
  • Structural information

Bradley MJ, Chivers PT, Baker NA. J Mol Biol, in
press.
25
Sequence conservation
DNA binding
Group 1.
Ni2
Ni2
Ni2
Group 3.
Ni2
Group 2.
Bradley MJ, Chivers PT, Baker NA. J Mol Biol, in
press.
26
Structural evidence
Bradley MJ, Chivers PT, Baker NA. J Mol Biol, in
press Schreiter ER, Wang SC, Zamble DB, Drennan
CL, PNAS 103(37), 13676-81, 2006.
27
NikR summary
  • Analysis of apo conformational ensemble
  • Global motions recapitulate X-ray structural
    changes
  • Contact correlations
  • Identify hubs of covariance in protein dynamics
  • Pathways of communication between NikR sites
  • Identified functionally-important residues
  • Some already implicated by experiment
  • Some currently undergoing testing

28
Application to thrombin allostery
  • Allosteric regulation by sodium cation binding
  • Shifts thrombin between pro- and anti-coagulant
    forms
  • Cation action possibly regulated by
    structurally-conserved water channel

Na binding site
Water channel
Active site
Exosite 1 fibrinogen fibrin thrombomodulin natural
inhibitor hirudin
Exosite 2 heparin (inhibitor) thrombomodulin
Zhang E, Tulinsky A, Biophys Chem 63, 185-200,
1997 Pineda AO, Carrell CJ, Bush LA, et al, J
Biol Chem, 279, 31842 - 53, 2004 Carrell CJ,
Bush LA, Mathews FS, Di Cera E, Biophys Chem,
121(3) 177-84, 2006.
29
Molecular dynamics of apo and holo thrombin
  • Two simulations
  • apo starting structure (1SGI)
  • holo starting structure (1SG8)
  • Simulation conditions
  • Periodic TIP3P water box
  • 200 mM NaCl (Kd of thrombin 200 mM)
  • NpT ensemble 298K, 1.0 atm
  • Amber 8.0 with the Amber99 forcefield
  • 115 ns per trajectory

30
Cation binding and unbinding events
  • Can classify states based on
  • Na binding several binding/unbinding events
    per trajectory
  • Water channel state water channel fills when
    Na is absent
  • How does Na binding and water channel state
    affect the active site?

31
Effects on active site
  • Rearrangement of hydrogen bonds in active site
    triad
  • Na binding improves catalytic competency
  • Water channel water competes for active site
    hydrogen bonds

32
Channel occupancy qualitative analysis
substate 1 5 waters 1SG8-bound 1SG8-unbound
substate 2 9 waters 1SG8-bound 1SG8-unbound
substate 3 11 waters 1SGI-unbound
33
Channel occupancy connectivity
  • Use contact probability analysis discussed
    before
  • Water channel
  • Protein
  • Entire system
  • Analyze spectrum of weighted graph Laplacian

Weighted adjacency matrix
Laplacian matrix
water channel cells
water channel cells
34
Channel occupancy connectivity
1SG8 unbound cluster 1 not connected 27 cells
1SG8 bound cluster 1 connection strength
0.0031 37 cells
1SG8 unbound cluster 2 connection strength
0.0166 42 cells
1SG8 bound cluster 2 connection strength
0.0259 46 cells
1SGI unbound connection strength 0.0544 48 cells
35
Communication with the active site
  • Use contact correlation clustering to assess
    communication between Na binding and active
    sites
  • apo strong, direct communication
  • holo weak, diffuse communication

1SGI-unbound
1SG8-bound
36
Identifying important residues
Residues in site definitions
Important residues identified by cluster analysis
Many residues already implicated experimentally.
37
Thrombin summary
  • Analysis of apo and holo conformational ensembles
  • Observation of a population shift of water
    channel associated with Na binding
  • Contact analyses demonstrate
  • Na binding disrupts water channel
  • Water channel coordinates neighboring residue
    motion
  • Water channel competes with active site hydrogen
    bonding
  • Initial results agree with some experiments
  • Still lots of work to be done!

Na
38
Overall summary
  • Methods to complement analyses of large-scale
    covariance and conformational change
  • Contact probability
  • Determines paths through proteins, water
    channels, etc.
  • Readily amenable to graph theoretic and related
    analyses
  • Useful for comparing simulations under different
    conditions (apo vs. holo)
  • Contact correlation
  • Identifies correlated fluctuations which can
    communicate information between sites
  • Assumes fluctuation-dissipation for analysis of
    allostery
  • Also defines graph structures
  • Tools available upon request

39
Acknowledgments
  • Collaborators
  • Peter Chivers (WUStL)
  • Enrico Di Cera (WUStL)
  • Students
  • Mike Bradley
  • Rachel Rice
  • Yong Huang
  • Todd Dolinsky
  • Funding
  • NAKFI
  • NSF (research, graduate fellowship)
  • NIH (training grant support)
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