Transcript PPT - AePIC
COMPARATIVE MOLECULAR DYNAMICS
SIMULATIONS TO STUDY ENZYMATIC COLD
ADAPTATION
Luca De Gioia
Molecular Modeling Laboratory,
Department of Biotechnology and Biosciences,
University of Milano-Bicocca, Italy
PSYCHROPHILIC ORGANISMS
• ARCTIC AND ANTARCTIC
½ Earth’s surface: oceans 1°C - 4°C
Deep sea – 1°C to 4°C
PSYCHROPHILIC ENZYMES: catalysis in extreme conditions
rational design of biocatalysts and biotechnological applications
Georlette et al, FEMS Microbiol. Rev., 28 (2004) 25.
PSYCHROPHILIC ENZYMES: an OVERVIEW
HIGH CATALYTIC EFFICIENCY at
0-30°C
THERMOLABILITY
STRUCTURAL FLEXIBILITY ?
• fewer intramolecular interactions
• more PROTEIN-SOLVENT interactions
Georlette et al, FEMS Microbiol. Rev., 28 (2004) 25.
Detailed INTRA-FAMILY structural comparisons
COMPARATIVE and
STATISTICAL
INVESTIGATIONS
• general features
• overlooking subtle structural
modifications
A general theory of enzymatic cold adaptation cannot
be formulated because…
Cold adaptation in different families is most probably obtained by
different EVOLUTIONARY STRATEGIES
Gianese, G., Bossa, F., Pascarella, S., Proteins, 47 (2002) 236.
Molecular dynamics
Proteins are not rigid molecules
•
•
•
•
Conformational changes
Protein folding
Molecular recognition (drug design)
Ion transport
The method is based on the Newton’s equation of motion:
Fi mi ai
Fi iV
d 2 ri
dV
mi 2
dri
dt
•(Numerical) integration of the equation of motion yields a trajectory.
•The average values of properties can be determined from the trajectory
MD shortcomings
• The integration step (dt) must be very small (1fs)
[supercomputing]
• The trajectory must be very long (to compute properties
the simulation must pass through all possible states
corresponding to the particular thermodynamic
constraints) [supercomputing]
MD protocol
To properly sample the phase space:
Multiple MD SIMULATIONS: Gromacs (50 ns, explicit solvent)
MD protocol
rmsd
1 N
2
(
r
r
)
i
0
N i1
Up to 10 ns
•Rmsd (mainchain)
N = number of atoms
r = position; r0 = initial position
•Protein gyration radius
•Total and potential energy
Elastases (serine protaeses)
3D STRUCTURE: 2 DOMAINS
antiparallel β-type fold (12 β-strands and 3
α-helices).
COLD-ADAPTED = atlantic salmon
elastase (SE)
MESOPHILIC = porcine elastase (PE)
FUNCTIONAL SITES: catalytic triad
(H57, D102 and S195) and specificity
pocket
Psychrophile/Mesophile comparison: primary sequences
Primary sequence (PE and SE,
~ 210-250 aa ): 68.2% identity
76 amino acidic substitutions (45
completely unrelated aa).
Maiale
Bovina
MerluzzoB
Salmone
Maiale
Bovina
MerluzzoB
Salmone
Maiale
Bovina
MerluzzoB
Salmone
VVGGTEAQRNSWPSQISLQYRSGSSWAHTCGGTLIRQNWVMTAAHCVDRELTFRVVVGEH
VVGGTAVSKNSWPSQISLQYKSGSSWYHTCGGTLIKQKWVMTAAHCVDSQMTFRVVLGDH
VVGGEDVRVHSWPWQASLQYKSGNSFYHTCGGTLIAPQWVMTAAHCIGSR-TYRVLLGKH
VVGGRVAQPNSWPWQISLQYKSGSSYYHTCGGSLIRQGWVMTAAHCVDSARTWRVVLGEH
**.* . ::** * ***
.. : *.***:*:
**:*****:.
:** :* *
NLNQ-NNGTEQYVGVQKIVVHPYWNTDDVAAGYDIALLRLAQSVTLNSYVQLGVLPRAGT
NLSQ-NDGTEQYISVQKIVVHPSWNSNNVAAGYDIAVLRLAQSATLNSYVQLGVLPQSGT
NMQDYNEAGSLAISPAKIIVHEKWD—-SSRIRNDIALIKLASPVDVSAIITPACVPDAEV
NLNT-NEGKEQIMTVNSVFIHSGWNSDDVAGGYDIALLRLNTQASLNSAVQLAALPPSNQ
.:
:
.:.:* *:
*:*::::
. .
: . :*
ILANNSPCYITGWGLTRTNGQLAQTLQQAYLPTVDYAICSSSSYWGSTVKNSMVCAGGDG
ILANNTPCYITGWGRTKTNGQLAQTLQQAYLPSVDYATCSSSSYWGSTVKTTMVCAGGDG
LLANGAPCYVTGWGRLWTGGPIADALQQALLPVVDHAHCSRYDWWGSLVTTSMVCAGGDG
ILPNNNPCYITGWGKTSTGGPLSDSLKQAWLPSVDHATCSSSGWWGSTVKTTMVCAGG-G
:*..
**:****
*.* . *:*. :
.: **
:*** : . *:*.** *
Comparative molecular dynamics (MD) simulations
Molecular flexibility is difficult to estimate experimentally but possibly
crucial to understand cold-adaptation
Comparative MD simulations
of proteins
•TIME-EVOLUTION of
MOLECULAR PROPERTIES
• evaluation of PROTEIN
FLEXIBILITY
Analysis of MD trajectories
Secondary Structure (SS) content
Hydrogen bonds
Solvent accessible surface
Psychrophile/Mesophile comparison: flexibility
• Identification of regions characterized by different flexibility in SE and PE
Root mean square
fluctuation (Rmsf) profiles:
highlight STRUCTURAL
FLEXIBILITY.
rmsf
1 N
2
(
r
r
)
i
N i1
N = number of atoms
r = position; <r> = average
position
Psychrophile/Mesophile comparison
Different RMSF
Amino acid COMPOSITION
LOCALIZATION on the 3D structure
Differences that could be related to cold adaptation
SE
PE
Insight obtained by MD simulations
• COLD-ADAPTED ELASTASES: localized flexibility
(proximity of catalytic site/specificity pocket).
• MESOPHILIC ELASTASES: scattered flexibility
(far from protein functional sites).
Design of “wet” experiments: site-directed mutagenesis
Papaleo, E., Fantucci, P., De Gioia L., J. Chem. Theory Comput., 1 (2005) 1286.
Trypsins (serine proteases)
• Specific for peptide cleavage at Lys and Arg sites
• Bind a Ca2+ ion
• Factors regulating autoproteolysis (genetic disorders)
MD investigation
Role of Ca2+ in structure stabilization and autolysis?
- The region K60-R117 (including the Ca2+
binding loop) can be a target for autolysis.
- Ca2+ has been proposed to induce an
autolysis-resistant conformation
- Autolysis in fish trypsins is less Ca2+ dependent
• Bovine and salmon trypsins
• Apo and holo forms
• Multiple MD simulations:
~ 200 ns
Investigation of autoproteolysis sites
• Effects due to Ca removal
Flexibility of R117 and K188 is enhanced in BT
Insight from MD simulations
• Ca2+ removal increases the flexibility of residues
forming the binding site, but…
• …it also leads to enhanced flexibility in remote
regions
• Ca2+ affects the flexibility of some autolysis sites
in bovine trypsin but not in salmon trypsin
(experimental data)
Design of “wet” experiments: site-directed mutagenesis
Papaleo E., Riccardi L., Villa C., Fantucci P., De Gioia L.,
Biochim. Biophys. Acta, 1764 (2006) 1397.
Acknowledgments
Department of
Biotechnology and
Biosciences, University of
Milano-Bicocca, Milano,
Italy
• Elena Papaleo
• Prof. Piercarlo Fantucci
• Chiara Villa, Laura Riccardi, Marco Pasi,
Rodolfo Gonella Diaza, Paolo Mereghetti,
Gianluca Santarossa
Department of Biochemistry,
University La Sapienza, Roma,
Italy
• Prof. Stefano Pascarella
• Giulio Gianese
• Daniele Tronelli
• Prof. Arne Smalas
Norwegian Structural Biology Centre,
Tromso University, Tromso, Norway
• Bjorn Bransdal
• Magne Olufsen