Modeling the Structural and Energetic basis for Functional Coupling in two Engineered Maltose Binding Protein/TEM-1 β-Lactamase Molecular Switches
Michael D. Daily1, Marc Ostermeier1,2, and Jeffrey J. Gray1,2
1Program
Abstract
Appropriate MBP
crystal structure
Emulate covalent bond(s) at junction(s) with
7-8Å distance constraint near junction
Appropriate BLA
crystal structure
Introduction
A
L-bound A
B
A
high-activity B
B
Interaction or covalent bond
Crude model
(starting structure)
Break junction(s), move MBP and BLA
apart; repack all sidechains
Ensemble of 5 repacked apart
structures
Calculate a Δscore between one repacked apart
structure and the ensemble-average energy of the
repacked complex.
This number should be
negative if complex formation is favorable.
For each structure the docking code generates, a
list of individual residue energies is calculated.
1omp.pdb
Large closing
motion upon
maltose binding
Two switches:
end-to-end
(MBP1-370)-(BLA24-286)
1.8
1.1
T164-165
(MBP1-165)-(BLA24-286)-(MBP164-370)
1.7
1.8
shift = 5
MBP-BLA
junction
• Search for energetic perturbations (changes in individual residue energies)
• Postulate switching mechanisms based on differences in the magnitude and
distribution of structural and energetic perturbations between maltose-unbound and
maltose-bound forms of a switch.
• Identify underlying structural principles of intra- and inter-molecular signal transduction.
-20.73
• Δscore of a docked structure correlates with free energy change of
interface formation.
• No significant differences in Δscore (and thus interface free energy)
between maltose – unbound and maltose-bound form of either structure
with perturbed E
end-end unbound
45.5
32.2
end-end bound
48.1
56.5
T164-165 unbound
36.0
69.2
T164-165 bound
22.7
26.3
End-to-end fusion:
Stabilized cluster
Destabilized cluster near
BLA active site may
reduce BLA activity in
maltose-unbound form
• A cluster of destabilized residues is present in the BLA active site in the
unbound form but not in the bound form, which may mean that the BLA
active site is more stable, and presumably more active, in the bound form
than in the unbound form.
BLA activity may be
closer to normal in
maltose-bound form
since active site is not
significantly destabilized
ΔE > 0 (destabilized)
ΔE < 0 (stabilized)
Destabilized
interfacial cluster
In both maltose-bound and maltose-unbound forms of the
T164-165 switch, only the MBP-BLA interface region shows
significant energetic perturbation
Bound MBP
Bound BLA
Bound BLA
Unbound MBP
Bound MBP
MBP-BLA junctions
MBP-BLA junctions
penicillin
Bound BLA
with perturbed rotamer
The sets of data that show rotamer shifts and energetic changes,
respectively, are about 30 residues per fusion protein. Thus, the
probability of these sets overlapping randomly is about (30/635)2, or
about 0.2%. 635 is the length of the fusion proteins.
ΔE represents the change in average energy from the repacked apart ensemble to the repacked complex
structure. Residues with |ΔE| > 0.7 are shown.
Maltose-unbound and maltose-bound forms of the end-to-end
switch have very different rotamer shift distributions
structure
Summary
maltose
Bound BLA
penicillin
T164-165 fusion:
• Small regions of BLA show more rotamer perturbations in the maltosebound form than in the maltose-unbound form.
• For both forms, a large majority of structural and especially energetic
perturbations are confined to the interfacial region; there are no significant
structural or energetic changes anywhere near the BLA active site.
• There was less consistency among docked structures (smaller clusters) for
both forms of this fusion than for the corresponding forms of the end-to-end
fusion
• Many residues near the junction had very high energies (100 or more),
indicating that the junction was poorly constructed.
• In the future, the junctions will be modeled explicitly using loop modeling
techniques rather than implicitly using distance constraints. Explicit modeling
of the junctions should reduce the wander space of BLA and produce
models that are more consistent (larger clusters of docked structures), and
thus more useful for detecting switching behavior.
General conclusions:
• Current modeling methodology can detect structural and energetic
changes far from an interface.
• These changes pass through only a small fraction of the protein (5-10%)
• Why does only this 5-10% transduce a signal?
Project goals:
• Search for structural perturbations (changes in sidechain rotamers)
-24.79
maltose
Maltose binding to MBP increases BLA activity from depressed to near-wild type.
• For both forms of each switch, examine how and where the structure is perturbed in
response to the creation of an MBP-BLA interface:
T164-165**
Bound MBP
penicillin
• Create computational models of both maltose-unbound and maltose-bound forms of
the two MBP/BLA switches from known structures of MBP and BLA.
-15.35
• BLA has significantly more of both rotamer perturbations and energetic
perturbations in the (maltose) unbound form of this switch than in the
(maltose) bound form.
2) Determine the ensemble-average energy and
standard deviation for that same residue in the
repacked apart structure.
maltose
-19.91
Only rotamers with shifts of three or greater are considered.
penicillin
Structural Comparison Results
kcat(no maltose) kcat/Km(no maltose)
end-to-end*
Rotamer and Energetic Perturbations show much greater
overlap than randomly expected.
Bound BLA
MBP-BLA
junction
Penicillin in BLA
active site
4) A residue is considered to have a significant
Δenergy if its Δenergy (absolute value) is greater
than 0.7 energy units and is larger than the
standard deviation of the average energy of both
repacked complex and repacked apart ensembles.
kcat/Km(maltose)/
sequence
shift = 4
Unbound MBP
Bound BLA
• A library of random insertions of BLA into MBP was screened over several steps
to isolate switches in which BLA activity changes when maltose binds MBP.
(Guntas & Ostermeier 2003). Two switches were found:
fusion
shift = 3
1) Determine the ensemble-average energy and
standard deviation for that residue in the repacked
complex .
Unbound MBP
kcat(maltose)/
shift = 2
BLA active site energetics differ between the maltose-bound and
maltose-unbound forms of the end-to-end switch
For each residue in the complex:
maltose
BLA hydrolyzes β –
lactam antibiotics like
penicillin above
Rotamer shifts:
Rotamer shifts between maltose-unbound (left) and maltose-bound (right) forms of the T164-165 fusion. The
repacked complex structures for the maltose-unbound and maltose-bound forms, respectively are shown.
Five repacked apart structures and five repacked complex structures are compared.
3) Subtract (2) from (1) to get a Δenergy for each
residue.
Upper lobe
1anf.pdb
regions of BLA with
significantly more rotamer
shifts in maltose-bound
form than in maltoseunbound form
Energetic comparisons
2) Determine how many times the MCR for
that residue occurs in the repacked apart
ensemble.
maltosebound
A Δscore of -20 corresponds to ~10 kcal/mol
Energetic Comparison Results
1) Determine most common Dunbrack
sidechain rotamer* (MCR) of that residue in
the repacked complex ensemble.
maltoseunbound
percent of perturbed E percent of perturbed rotamers
Ensemble of 5 repacked
complex structures
penicillin
1fqg.pdb
Lower lobe of MBP
interacts with BLA
only in the maltoseunbound form
Repack all sidechains in the complex,
but leave backbone in place
Bound BLA
Lower lobe
Interface Δscores for both forms of MBP-BLA and T164-165
Errors in Δscore are 5-10 score units.
interface is small and
loosely packed in both
maltose- unbound and
maltose-bound forms.
Backbone of MBP-BLA complex
Switch components: Maltose Binding Protein
(MBP) and TEM-1 β-lactamase (BLA):
Bound MBP
penicillin
maltose
Connect junctions using loop
modeling and/or energy minimization
Docking program
*Dunbrack and Cohen (1997).
Unbound MBP
Bound BLA
penicillin
Select top-scoring structure of largest cluster
4) For the a residue in one of the MBP-BLA
complexes, a rotamer shift of 3 or greater is
considered significant.
• Ligand (L) binds to protein A and alters the catalytic activity of protein B
• Many natural protein/protein switches exist
Bound MBP
Cluster top 200 structures
3) Subtract (2) from (1) to get the rotamer
shift for that residue. The rotamer shift can
range from -4 to 5.
L
Bound BLA
Differences in interface free energy between maltoseunbound and maltose-bound forms probably do not explain
switching in either the end-to-end or T164-165 swtiches.
Generate 5000 docked structures
Manual molecular modeling
For each residue in the complex:
A hypothetical ligand-binding controlled molecular switch
Unbound MBP
Dock perturbation run
Rotamer comparisons
low-activity B
Significant rotamer shifts in both maltose-unbound and
maltose-bound forms of the T164-165 (internal) fusion are
primarily interfacial
Methods
In molecular switching, the recognition of an external signal such as
ligand binding by one protein is coupled to the catalytic activity of a second
protein.
Many natural molecular switches exist, but novel engineered
molecular switches that can couple a chosen external signal to a chosen
chemical activity could be of great use in manipulating and controlling
biological systems. Recently, Ostermeier and colleagues have used directed
evolution to engineer two insertions of TEM-1 β-lactamase (BLA) into maltose
binding protein (MBP) in which the binding of maltose to MBP increases the
rate of BLA hydrolysis of nitrocefin, a substrate analogue. BLA is inserted
internally at position 165 of MBP in one of these molecular switches and at the
end of MBP in the other switch. To investigate the biophysical basis for the
transfer of the maltose binding signal from the maltose-binding site of MBP to
the BLA active site, computational models of the two switching proteins are
being constructed and analyzed. Recently developed protein folding and
docking programs have been used to construct models of both maltoseunbound and maltose-bound forms of each switch. These models have been
used to identify residues in both forms of each switch that undergo significant
structural and/or energetic changes as a result of the creation of an MBP/BLA
interface. For both forms of the end-to-end fusion, significant structural and/or
energetic changes have been identified in multiple residues in MBP and/or BLA
that are distant from the interface. Most interestingly, a cluster of residues at
the BLA active site is predicted to increase in stability when maltose binds
MBP, which could cause BLA activity to correspondingly increase. For both
forms of the internal fusion, almost all significant structural changes are
confined to the MBP/BLA interface, which one would expect if this fusion did
not display switching behavior. Current modeling techniques will have to be
improved to generate models for both forms of T164-165 that are accurate
enough to detect possible switching mechanisms. The possible switching
mechanisms that are derived from these models may contribute to a greater
knowledge of how signals are transduced between sites in individual proteins
and protein complexes.
Unbound A
in Molecular Biophysics and 2Department of Chemical and Biomolecular Engineering, Johns Hopkins University
penicillin
Interface has a
significant void
BLA has multiple rotamer
shifts of three or more far
from the interface, but only in
the maltose-unbound form
Possible experiments:
highly
complementary
interface
Rotamer shifts:
Unexpected cluster of
significant rotamer shifts
present only in the
maltose-bound form
shift = 2
shift = 3
shift = 4
shift = 5
Rotamer shifts between maltose-unbound (left) and maltose-bound (right) forms of the end-to-end fusion. The
repacked complex structures of maltose-unbound and maltose-bound forms, respectively, are shown. Five
repacked apart structures and five repacked complex structures are compared.
Energetic perturbations do
not propagate far from the
interface
Energetic perturbations are
localized to the interface
ΔE > 0 (destabilized)
ΔE < 0 (stabilized)
• Attempt to alter the coupling between MBP and BLA in the end-to-end
fusion by mutating residues in the destabilizing cluster near the active site.
References
Guntas, G. and Ostermeier, M. (2003) Creation of an Allosteric Enzyme by Domain Insertion.
Submitted.
Gray, J. J. et al. (2003). Protein-Protein Docking with Simultaneous Optimization of Rigid-Body
Displacement and Side-chain Conformations. J. Mol. Biol., in press.
ΔE represents the change in average energy from the repacked apart ensemble to the
repacked complex structure. Residues with |ΔE| > 0.7 are shown.
Dunbrack, R.L., and Cohen, F.E. (1997). Bayesian Statistical Analysis of Protein Sidechain
Rotamer Preferences. Protein Sci. 6: 1661-1681.