Introduction

1. Molecular dynamics simulations and membrane protein structure quality Anthony Ivetac and Mark S. P. Sansom European Biophysics Journal, Volume 37, Number 4, April 2008Tim NugentBUGF 11/09/08

2. Introduction • The PDB now contains ∼120 unique membrane protein structures, about 1% of the total deposited structures. • However, it should be recalled that the majority of membrane protein structures are determined in a non-membrane environment. • Given the key roles of lipids in function, this may be an important limitation. Furthermore, a significant number of the membrane protein structures determined are at relatively low resolutions. • Thus, computational tools to evaluate the “quality” of a membrane protein should play a key role in membrane protein structural biology. • This is also the case for assessment of, e.g. homology models of human membrane proteins based on structures of (distant) bacterial homologues. • Standard methods for measuring protein quality provide an assessment of local stereochemistry but do not consider the global fold of a membrane protein model. • Molecular dynamics (MD) simulations in a lipid bilayer environment provide a possible tool to address the latter aspect.

3. An unusual scenario • A structure is available where the TM helices have been correctly assigned, but where the packing together of the TM helices is incorrect. • The 4.2 Å resolution structure for a bacterial ABC transporter [MsbA from S. typhimurium] has proved to be incorrect in both the handedness of the structure and the topology. • The TM helices within the (incorrect) model agree well with a consensus of sequence based predictions. • Such a situation could conceivably also arise, e.g. either via homology modelling with a low sequence identity between target and template, via restraint-directed fitting of a TMH fold to low resolution electron density data, or via modelling of an α-helical membrane protein to NMR data. • In this paper two MD simulations are compared: one of the MsbA model structure and one of another ABC protein, the vitamin B12 transporter BtuCD, each protein embedded in a dimyristoylphosphatidlycholine (DMPC) bilayer. • Comparative analyses of structural integrity of the protein in the two simulations demonstrate that MD simulations may be used as a measure of quality of a membrane protein structure.

4. Structure of the ABC Transporter MsbA in Complex with ADP·Vanadate and LipopolysaccharideChristopher L. Reyes and Geoffrey Chang Retraction An in-house data reduction program introduced a change in sign for anomalous differences. This program, which was not part of a conventional data processing package, converted the anomalous pairs (I+ and I-) to (F- and F+), thereby introducing a sign change. As the diffraction data collected for each set of MsbA crystals and for the EmrE crystals were processed with the same program, the structures reported in (1-3, 5, 6) had the wrong hand. The error in the topology of the original MsbA structure was a consequence of the low resolution of the data as well as breaks in the electron density for the connecting loop regions. Unfortunately, the use of the multicopy refinement procedure still allowed us to obtain reasonable refinement values for the wrong structures. 1. G. Chang, C. B. Roth, Science 293, 1793 (2001). 2. C. L. Reyes, G. Chang, Science 308, 1028 (2005). 3. O. Pornillos, Y.-J. Chen, A. P. Chen, G. Chang, Science 310, 1950 (2005). 1JSQ, 1PF4, and 1Z2R for MsbA and 1S7B and 2F2M for EmrE have been moved to the archive of obsolete PDB entries.

5. Simulation Methods • Molecular dynamics simulations were carried out with GROMACS using the GROMOS96 force-field. • Simulations were run in the NPT ensemble at 310 K and 1 atmosphere. Each system was equilibrated (for 0.5 ns for MsbA and for 0.2 ns for the intact BtuCD structure). • During equilibration the Berendsen thermostat and Berendsen barostat algorithms were employed, while the 20 ns production runs employed the Nosé–Hoover thermostat and Parrinello–Rahman barostat algorithms. • Long-range electrostatic interactions were calculated with the particle mesh Ewald (PME) method. All bonds were constrained using the LINCS algorithm, with the use of a 2 fs timestep. • Analysis was carried out using the GROMACS suite of analysis programs.

6. Results • MsbA simulation reveals significant structural deformations in both the transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) of the model. • The protein appears to “collapse” and the TMDs exhibit a striking loss of symmetry. • The transmembrane helices (TMHs) undergo significant movements from their starting positions. • The NBDs appear to retain their fold better. • In contrast, the BtuCD structure exhibits little change during the course of the 20 ns simulation.

7. Conformational drift • Calculated RMSD of all Cα atoms over 20 ns. • In the MsbA simulation, there is an initial (over the first 1 ns) steep increase in the RMSD for all residues of the protein to ∼6 Å, which is followed by a steady increase to a value of ∼9 Å at 20 ns. • In contrast, for the BtuCD simulation there is an initial (over the first 0.1 ns) rise to ∼2 Å, and then the value plateaus, with a final Cα RMSD of ∼2.7 Å after 20 ns. • For the TMDs, it is clear that almost all of the conformational drift is the result of intra-TMD deformations. • For the NBDs, the situation is a little more complex. Although the intra-NBD RMSDs are high, it is evident that inter-NBD motions also make a contribution to the total conformational drift of this region of the protein. • Thus, the major “instability” in the MsbA fold would seem to be in the transmembrane domain.

8. Helix retention • Measured the extent to which those TMD residues present in an α-helix retain this secondary structure during a simulation. • In the MsbA simulation, 15% of these residues lose their α-helicity • In the BtuCD simulation the comparable loss is only 1%. • Visualization of the TMDs from MsbA at the end of the simulation reveals both truncation of and breaks in the α-helices. • In contrast, for the TMDs of BtuCD, all α-helices remain intact at the end of the simulation.

9. Secondary structure analysis Secondary structure analysis (using DSSP) of TMDs of (a) MsbA and (b) BtuCD. In each case, the secondary structure at start of each simulation (Start) is compared with the dominant secondary structure (Dom). Position of the transmembrane helices of TMD1 and of TMD2 is indicated by green and red bars, respectively (blue α-helix; yellow turn; black β-bridge; purple 5-helix; dark grey 310-helix; light grey coil). • Comparison for the two simulations reveals a marked loss of α-helicity for most of the 12 TMHs of MsbA. • These changes are significant given that the location of the TMHs within the initial MsbA model is in good agreement with the consensus predicted locations of TMHs in the MsbA sequence.

10. Orientation and interaction with respect to the lipid bilayer • The tilt angle of the long axis of the protein relative to the bilayer normal was calculated. • For MsbA there was a significant change over 20 ns, with the tilt angle initially ∼0° but progressively shifting to ∼22° over the course of the simulation. • In contrast that for BtuCD fluctuates within 0° to 5° over the course of the simulation.

11. Lipid bilayer behaviour • Monitored the behaviour of the lipid bilayer in the two simulations. • The bilayer in the MsbA simulation shows considerable local deformation relative to a control simulation of a DMPC bilayer without an inserted protein. • In contrast, for BtuCD the thickness of the bilayer remains identical to that in the control DMPC simulation.

12. Conclusions • These simulations suggest that MD can be used to detect an incorrect membrane protein structure or model as one which exhibits considerable conformation instability during such simulations. • MD simulation could be a valuable quality control tool for membrane protein structural biology. • A single 20 ns simulation of a protein of comparable size to MsbA takes ∼480 CPU-days, i.e. ∼1 month on 16 CPUs. • Study could be extended to a wider range of membrane proteins, and to more subtle differences/errors in structure. • One way in which this could be done would be to consider those membrane proteins for which multiple structures at a range of resolutions are available. • In more general terms, the simulations described above suggest that one may use MD simulations to detect incorrect packing of otherwise correctly predicted TM α-helices in a membrane protein model.