Biophysical and computational studies of membrane penetration by the GRP1 pleckstrin homology domain - PubMed (original) (raw)

Biophysical and computational studies of membrane penetration by the GRP1 pleckstrin homology domain

Craig N Lumb et al. Structure. 2011.

Abstract

The pleckstrin homology (PH) domain of the general receptor for phosphoinositides 1 (GRP1) exhibits specific, high-affinity, reversible binding to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) at the plasma membrane, but the nature and extent of the interaction between this bound complex and the surrounding membrane environment remains unclear. Combining equilibrium and nonequilibrium molecular dynamics (MD) simulations, NMR spectroscopy, and monolayer penetration experiments, we characterize the membrane-associated state of GRP1-PH. MD simulations show loops flanking the binding site supplement the interaction with PI(3,4,5)P(3) through multiple contacts with the lipid bilayer. NMR data show large perturbations in chemical shift for these loop regions on binding to PI(3,4,5)P(3)-containing DPC micelles. Monolayer penetration experiments and further MD simulations demonstrate that mutating hydrophobic residues to polar residues in the flanking loops reduces membrane penetration. This supports a "dual-recognition" model of binding, with specific GRP1-PH-PI(3,4,5)P(3) interactions supplemented by interactions of loop regions with the lipid bilayer.

Copyright © 2011 Elsevier Ltd. All rights reserved.

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Graphical abstract

Figure 1

Figure 1

Structural Features of GRP1-PH (A) The three binding loops, β1/β2 (black), β3/β4 (green), and β6/β7 (blue), and their positions relative to the I(1,3,4,5)P4 head group. (B) The geometry of the I(1,3,4,5)P4 binding site, with the key amino acid residues referred to in the text shown as stick representations in pink. (C) The initial setup for the MD simulations, with the centers of the phosphate groups shown as gray spheres and the lipid bilayer depicted as a translucent white surface. Water molecules and ions are omitted for clarity. In all cases, the protein is shown as a ribbon diagram, and the I(1,3,4,5)P4 head group is shown as a stick model.

Figure 2

Figure 2

Membrane Penetration of GRP1-PH Observed during the MD Simulations (A) Mean number of nonpolar protein-lipid contacts per residue per ps over the 100 ns for the two wild-type protein simulation (one simulation in black and the second simulation in blue). (B) The same measure for the two A346E mutant simulations. (C) and (D) show these contacts projected on to the molecular surface of the protein for the wild-type and A346E mutants respectively, illustrating the decrease in penetration depth of the β6/β7 loop upon mutation. The color scale corresponds to the plots in (A) and (B) and shows the mean number of nonpolar protein-lipid contacts per residue per ps, varying from 0 (blue) to 2 (red). Nonpolar contacts are defined as the number of POPC tail carbon atoms within 4 Å of a protein heavy atom. See also Figure S3.

Figure 3

Figure 3

Association of the PI(3,4,5)P3-Bound GRP1-PH with Membrane-Mimicking DPC Micelles Monitored by NMR (A) Superimposed 1H, 15N HSQC spectra of the PI(3,4,5)P3 (0.4 mM)-bound PH domain (0.2 mM) in the DPC-free state (black) and in the presence of 5.1 mM DPC micelles (red) collected at pH 6.8. (B) The histograms show normalized (Grzesiek et al., 1996) chemical shift changes induced in the backbone amides of the PI(3,4,5)P3-bound GRP1 PH domain by DPC micelles at indicated pH. Significant changes in resonances are judged to be greater than the average plus one standard deviation (red line). (C) Residues that display significant changes in chemical shift are colored in red and pink for large and medium changes, respectively. Mutated residues are orange. The head group of PI(3,4,5)P3 is shown as a stick model and colored green.

Figure 4

Figure 4

Simulation Snapshots of GRP1-PH Being Pulled from the Bilayer Surface (A) Snapshot at t = 0 ns for the neutral H355 case. (B) Snapshot at t = 36 ns, again for the neutral H355 case. The interaction between the β6/β7 loop and the membrane lipids is sufficiently strong to completely remove a lipid from the bilayer (shown in yellow) at this pulling velocity, while another lipid (shown in red) is partially extracted. See also Figure S4.

Figure 5

Figure 5

Potential Energy of the Protein-Membrane Complex over the SMD Simulations Short-range components of the van der Waals and electrostatic contributions to the potential energy are shown for the slower SMD simulations, of duration 80 ns and a pulling rate of 0.5 Å/ns. (A) van der Waals potential energy and (B) electrostatic potential energy for the SMD simulation with H355 in a neutral state. (C) van der Waals potential energy and (D) electrostatic potential energy for the SMD simulation with H355 in a protonated state. See also Figures S1 and S2.

Figure 6

Figure 6

Monolayer Penetration Experiments of GRP1-PH and Respective Mutations Insertion of the wild-type GRP1 PH domain (filled circles), V278E (filled triangles), Y298E (filled squares), A346E (filled diamonds), and V351E (open squares) mutations into a POPC/POPE/PI(3,4,5)P3 (77:20:3) monolayer in a subphase of 10 mM HEPES/0.16 M KCl (pH 7.4) monitored as a function of π.

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