Michiel J. M. Niesen

Thermostabilization of the β1-Adrenergic Receptor Correlates with Increased Entropy of the Inactive State

The dynamic nature of GPCRs is a major hurdle in their purification and crystallization. Thermostabilization can facilitate GPCR structure determination, as has been shown by the structure of the thermostabilized β1-adrenergic receptor (β1AR) mutant, m23-β1AR, which has been thermostabilized in the inactive state. However, it is unclear from the structure how the six thermostabilizing mutations in m23-β1AR affect receptor dynamics. We have used molecular dynamics simulations in explicit solvent to compare the conformational ensembles for both wild type β1AR (wt-β1AR) and m23-β1AR. Thermostabilization results in an increase in the number of accessible microscopic conformational states within the inactive state ensemble, effectively increasing the side chain entropy of the inactive state at room temperature, while suppressing large-scale main chain conformational changes that lead to activation. We identified several diverse mechanisms of thermostabilization upon mutation. These include decrease of long-range correlated movement between residues in the G-protein coupling site to the extracellular region (Y227A(5.58), F338M(7.48)), formation of new hydrogen bonds (R68S), and reduction of local stress (Y227(5.58), F327(7.37), and F338(7.48)). This study provides insights into microscopic mechanisms underlying thermostability that leads to an understanding of the effect of these mutations on the structure of the receptor.

Interaction of actin with carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) receptor in liposomes is Ca2+- and phospholipid-dependent.

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749–5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca2+. In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca2+ ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of 13C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca2+ dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca2+-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.

Structurally detailed coarse-grained model for Sec-facilitated co-translational protein translocation and membrane integration

We present a coarse-grained simulation model that is capable of simulating the minute-timescale dynamics of protein translocation and membrane integration via the Sec translocon, while retaining sufficient chemical and structural detail to capture many of the sequence-specific interactions that drive these processes. The model includes accurate geometric representations of the ribosome and Sec translocon, obtained directly from experimental structures, and interactions parameterized from nearly 200 μs of residue-based coarse-grained molecular dynamics simulations. A protocol for mapping amino-acid sequences to coarse-grained beads enables the direct simulation of trajectories for the co-translational insertion of arbitrary polypeptide sequences into the Sec translocon. The model reproduces experimentally observed features of membrane protein integration, including the efficiency with which polypeptide domains integrate into the membrane, the variation in integration efficiency upon single amino-acid mutations, and the orientation of transmembrane domains. The central advantage of the model is that it connects sequence-level protein features to biological observables and timescales, enabling direct simulation for the mechanistic analysis of co-translational integration and for the engineering of membrane proteins with enhanced membrane integration efficiency.

Improving membrane protein expression by optimizing integration efficiency

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies.

Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon

During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide experiments and coarse-grained molecular dynamics to measure and elucidate the molecular origins of forces that are exerted on NCs during cotranslational membrane insertion and translocation via the Sec translocon. The approach enables deconvolution of force contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a readout of conformational changes in the Sec translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis.

Inversion of Signal Sequence Topology during Membrane Integration

In many cases, the topology of membrane proteins is established during co-translational membrane integration. This process involves the Sec translocon, a heterotrimeric protein-conducting channel that allows for both the translocation of secreted domains across the membrane through the central pore and the integration of membrane domains directly into the lipid bilayer through a lateral opening. For many proteins, the N-terminus is retained on the cytosolic side of the membrane and forms an inverted (type II) topology that threads the C-terminus through the channel. This inverted topology is hypothesized to involve a head-first intermediate which then undergoes a step-wise inversion process to its final topology. We use a newly developed coarse-grained model to simulate the integration of the signal sequence during the elongation of the nascent chain on the minute-long timescales that are relevant to the biological process. This coarse-grained simulation method enables direct comparisons to experimentally measured energetics of ribosome-nascent chain to translocon interactions. We observe a series of pulling and pushing forces on the ribosome-nascent chain as translation proceeds and identify a head-first intermediate whose inversion is driven by the entropic confinement of nascent chain residues in the ribosome-translocon junction.

Role of water and G Protein in modulating agonist affinity in GPCRs

Predicting accurate ligand poses and ligand selective receptor conformations are imperative in designing efficacious, functionally specific drugs for G-protein coupled receptors. Comparison of the crystal structures of carazolol (inverse agonist), formoterol(agonist), and agonist with G-protein mimic bound β2 Adrenergic Receptor (β2AR) shows that the agonist stabilizes a slightly different conformation when there is no G-protein bound. The G-protein bound receptor state exhibits a high affinity conformation for the agonist. Using the computational method LITiCon, we have calculated the activation pathways for full, partial and inverse agonists of β2AR, which are in agreement with fluorescence intensity lifetime measurements. MD simulations starting from various conformations along the activation pathway (total ∼1.5 μs) show that in the absence of G protein, norepinephrine (agonist) stabilizes an intermediate receptor state that is similar to the formoterol bound intermediate state without the G-protein, in agreement with the recent crystal structure of formoterol bound β2AR. Thus coupling to G protein may be needed for stabilizing the fully active state.Using Liticon method we have calculated the activation pathway of agonist bound adenosine receptor A2A starting from its inactive state. We found that water plays a important role in the docking of the antagonist as well as the agonist.We recently predicted the structures of D3 dopamine receptor and CXCR4 chemokine receptor as part of the competition for the assessment of computational methods in GPCR modeling (GPCR Dock 2010). The predicted docked poses of both the ligands are in close agreement with the crystal structures. The details of the methods and comparison with the crystal structures will be presented.