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 maltose-unbound 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.

Modeling the Structural and Energetic basis for Functional Coupling in two Engineered Maltose Binding Protein/TEM-1 β-Lactamase Molecular SwitchesMichael D. Daily1, Marc Ostermeier1,2, and Jeffrey J. Gray1,2

1Program in Molecular Biophysics and 2Department of Chemical and Biomolecular Engineering, Johns Hopkins University

L

Unbound A low-activity B L-bound A high-activity B

A B A B

Interaction or covalent bond

• Ligand (L) binds to protein A and alters the catalytic activity of protein B• Many natural protein/protein switches exist

Abstract

IntroductionA hypothetical ligand-binding controlled molecular switch

Switch components: Maltose Binding Protein (MBP) and TEM-1 β-lactamase (BLA):

Two switches:

• 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:

Project goals:

• Create computational models of both maltose-unbound and maltose-bound forms of the two MBP/BLA switches from known structures of MBP and BLA.

• For both forms of each switch, examine how and where the structure is perturbed in response to the creation of an MBP-BLA interface:

• Search for structural perturbations (changes in sidechain rotamers)

• 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.

Methods

Structural Comparison Results

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.

Maltose-unbound and maltose-bound forms of the end-to-end switch have very different rotamer shift distributions

Significant rotamer shifts in both maltose-unbound and maltose-bound forms of the T164-165 (internal) fusion are primarily interfacial

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.

• 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

ΔE > 0 (destabilized)

ΔE < 0 (stabilized)

Bound BLA

Bound MBP

Penicillin in BLA active site

penicillin

maltose

Stabilized clusterDestabilized cluster near BLA active site may reduce BLA activity in maltose-unbound form

Unbound MBPBound BLA

BLA activity may be closer to normal in maltose-bound form since active site is not significantly destabilized

MBP-BLA junction

MBP-BLA junction

Destabilized interfacial cluster

BLA active site energetics differ between the maltose-bound and maltose-unbound forms of the end-to-end switch

Bound BLA Unbound MBP Bound BLA Bound MBP

penicillin

MBP-BLA junctionsMBP-BLA junctions

Energetic perturbations do not propagate far from the interface

Energetic perturbations are localized to the interface

ΔE > 0 (destabilized)

ΔE < 0 (stabilized)

Bound BLA

Bound BLABound BLA

Bound BLA

Bound BLA

Unbound MBP

Unbound MBP

Unbound MBP Bound MBP

Bound MBP

Bound MBP

In both maltose-bound and maltose-unbound forms of the T164-165 switch, only the MBP-BLA interface region shows significant energetic perturbation

Rotamer shifts:

shift = 2 shift = 3

shift = 4 shift = 5

penicillin

penicillin

maltose

highly complementary interface

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

Unexpected cluster of significant rotamer shifts present only in the maltose-bound form

Rotamer shifts:

shift = 2 shift = 3

shift = 4 shift = 5

interface is small and loosely packed in both maltose- unbound and maltose-bound forms.

Lower lobe of MBP interacts with BLA only in the maltose-unbound form

regions of BLA with significantly more rotamer shifts in maltose-bound form than in maltose-unbound form

Differences in interface free energy between maltose-unbound and maltose-bound forms probably do not explain switching in either the end-to-end or T164-165 swtiches.

maltose-unboundmaltose-bound

end-to-end* -19.91 -15.35

T164-165** -24.79 -20.73

Interface Δscores for both forms of MBP-BLA and T164-165

Errors in Δscore are 5-10 score units.

A Δscore of -20 corresponds to ~10 kcal/mol

penicillin

penicillin

penicillin

maltose

maltose

Appropriate MBP crystal structure

Appropriate BLA crystal structure

Crude model (starting structure)

Manual molecular modeling

Docking program

Break junction(s), move MBP and BLA apart; repack all sidechains

Ensemble of 5 repacked apart structures

Emulate covalent bond(s) at junction(s) with 7-8Å distance constraint near junction

Dock perturbation run

Generate 5000 docked structures

Cluster top 200 structures

Select top-scoring structure of largest cluster

Backbone of MBP-BLA complex

Connect junctions using loop modeling and/or energy minimization

Repack all sidechains in the complex, but leave backbone in

place

Ensemble of 5 repacked complex structures

Rotamer comparisons Energetic comparisons

For each residue in the complex:

1) Determine most common Dunbrack sidechain rotamer* (MCR) of that residue in the repacked complex ensemble.

2) Determine how many times the MCR for that residue occurs in the repacked apart ensemble.

3) Subtract (2) from (1) to get the rotamer shift for that residue. The rotamer shift can range from -4 to 5.

4) For the a residue in one of the MBP-BLA complexes, a rotamer shift of 3 or greater is considered significant.

*Dunbrack and Cohen (1997).

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.

For each residue in the complex:

1) Determine the ensemble-average energy and standard deviation for that residue in the repacked complex .

2) Determine the ensemble-average energy and standard deviation for that same residue in the repacked apart structure.

3) Subtract (2) from (1) to get a Δenergy for each residue.

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.

1omp.pdb

1anf.pdb

1fqg.pdb

Upper lobe

Lower lobe

maltose

penicillin

Large closing motion upon maltose binding

BLA hydrolyzes β – lactam antibiotics like penicillin above

Energetic Comparison Results

Summary

End-to-end fusion:• 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.

• 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.

T164-165 fusion:• Small regions of BLA show more rotamer perturbations in the maltose-bound 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?

Possible experiments:• 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.

Rotamer and Energetic Perturbations show much greater overlap than randomly expected.

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.

Only rotamers with shifts of three or greater are considered.

ΔE represents the change in average energy from the repacked apart ensemble to the repacked complex structure. Residues with |ΔE| > 0.7 are shown.

ΔE represents the change in average energy from the repacked apart ensemble to the repacked complex structure. Residues with |ΔE| > 0.7 are shown.

ReferencesGuntas, 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.

Dunbrack, R.L., and Cohen, F.E. (1997). Bayesian Statistical Analysis of Protein Sidechain Rotamer Preferences. Protein Sci. 6: 1661-1681.

percent of perturbed E percent of perturbed rotamers

structure with perturbed rotamer 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

kcat(maltose)/ kcat/Km(maltose)/

fusion sequence kcat(no maltose) kcat/Km(no maltose)

end-to-end (MBP1-370)-(BLA24-286) 1.8 1.1

T164-165 (MBP1-165)-(BLA24-286)-(MBP164-370) 1.7 1.8

Maltose binding to MBP increases BLA activity from depressed to near-wild type.