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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 i1
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 i1
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