Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

doi:10.1016/j.jsb.2009.09.009

Journal of Structural Biology

Volume 169, Issue 1, January 2010, Pages 116-123

https://doi.org/10.1016/j.jsb.2009.09.009 Get rights and content

Abstract

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g− conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Oγ atom of Ser and Thr and the backbone carbonyl oxygen at position i − 3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the β₂-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.

Introduction

While roughly 15–30% of the eukaryote genes encode membrane proteins (Fleishman et al., 2006), less than 2% of the structures deposited at the Protein Data Bank correspond to this class (http://pdbtm.enzim.hu/) (Tusnady et al., 2005). Transmembrane (TM) α-helical proteins constitute the majority of integral membrane proteins (Fleishman et al., 2006). In addition to canonical α-helices, these TM bundles are also formed by non-canonical π-like helices, 3₁₀-like helices and kinked helices (Rigoutsos et al., 2003). Predicting and quantifying distortions within TM segments is important for understanding processes as diverse as ligand recognition, protein function, and protein folding (Deupi et al., 2007, Bowie, 2005). Helix distortions are most commonly induced by Pro, which can be identified with more than 90% reliability simply by looking at Pro abundance in a multiple sequence alignment (Yohannan et al., 2004). However, it has been shown that other residues are also able to induce distortions in the structure of TMs (Rigoutsos et al., 2003). For instance, we have previously shown that both Ser and Thr residues, either alone (Ballesteros et al., 2000) or in combination with Pro (Govaerts et al., 2001, Deupi et al., 2004), also induce distinctive distortions in TMs. Membrane proteins, thus, incorporate in the sequence of their TMs specific residues like Pro, Gly, Ser, and Thr (Senes et al., 2000), introducing a flexible point and assisting in helix movements (Sansom et al., 2000) or stabilizing local regions of structural relevance.

In addition to its role in modulating the structure of TMs, Ser and Thr residues are also reported to play key roles in preserving the overall structure of membrane proteins (Lopez-Rodriguez et al., 2002), stabilizing the interactions between TMs (Dawson et al., 2002, Eilers et al., 2002), and regulating protein function (Munshi et al., 2003, Jongejan et al., 2005, Pellissier et al., 2009). In addition, mutations involving polar residues in TM segments are often associated with protein malfunction (Partridge et al., 2004, Smit et al., 2007), being the most common disease-causing mutations in membrane proteins (Joh et al., 2008).

These roles of Ser and Thr are ultimately encoded in the chemical properties of their polar side chains. The short side chains of Ser and Thr have a limited rotamer conformational space. The gauche− (g−), gauche+ (g+), and trans (t) staggered conformations are strongly preferred relative to the eclipsed conformations. The only exception is the t conformation of Thr, which is unfavorable due to the steric clash of the side chain methyl group with the backbone carbonyl at the i − 3 position (McGregor et al., 1987). Ser and Thr in g+ or g− are capable to hydrogen bond the backbone carbonyl in the previous turn of the helix (McGregor et al., 1987).

We have previously shown that the g− conformation of Ser and Thr residues modifies the conformation of Ser/Thr-containing α-helices (Ballesteros et al., 2000). This conclusion was achieved from statistical analysis of crystal structures of mostly soluble proteins, with only four structures of membrane proteins included in the analysis. It has been suggested that the membrane environment considerably perturbs the rotamer frequencies compared to soluble proteins (Chamberlain and Bowie, 2004). Thus, the present study aims to provide additional insight into the structural consequences of the different rotamer conformations of Ser and Thr on the overall geometry of TMs, combining two complementary sets of structural data: a database of helix stretches extracted exclusively from crystal structures of membrane proteins, and ensembles of model TMs generated by molecular dynamics (MD) simulations in an explicit hydrophobic environment.

Section snippets

Database of transmembrane helix segments

The atomic coordinates of the membrane proteins listed in Table 1 were extracted from the RCSB Protein Data Bank (Berman et al., 2000). The coordinates of all TM stretches with a length of 12 residues and bearing Ala (as a control), Ser or Thr in the 8th position were extracted for analysis. Selection of longer stretches would have lead to a reduction of the sample size. Only stretches with Ala/Ser/Thr exposed to the membrane were kept for analysis, in order to avoid interfering effects from

Influence of Ser and Thr side chain conformation in the structure of the helix backbone

Table 2 summarizes the mean and standard deviation of the backbone φ and ψ dihedral angles of Ser and Thr residues in their possible rotamer conformations and of Ala (control) calculated from the experimental structures extracted from the Protein Data Bank (i.e. Ala^PDB, Ser^PDBg−, Thr^PDBg−, Ser^PDBg+, Thr^PDBg+, Ser^PDBt, and Thr^PDBt). We have previously shown that there is a clear influence of the environment on the main chain conformation (φ and ψ dihedral angles) of α-helices (Olivella et al.,

Conclusions

Ser and Thr are the most prevalent polar residues in TMs, each constituting 5% of the residues (Senes et al., 2000). These amino acids, unlike other polar or charged residues, do not destabilize TMs (Monne et al., 1999), as their hydrogen bonding potential can be satisfied by interacting with the carbonyl oxygen in the preceding turn of the same helix (Gray and Matthews, 1984) (Fig. 1). To assess the influence of this side chain-backbone hydrogen bond interaction in the structure of TMs, we

Acknowledgments

This work has been supported by grants from the Ministerio de Educación y Ciencia (Spain) (MEC, SAF2006-04966, SAF2007-67008) and Instituto de Salud Carlos III (RD07/0067/0008). X.D. is supported by MEC through the Ramon y Cajal program.

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¹: Present address: Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany.

²: These authors contributed equally to this work.

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Influence of the g− conformation of Ser and Thr on the structure of transmembrane helices

Abstract

Introduction

Section snippets

Database of transmembrane helix segments

Influence of Ser and Thr side chain conformation in the structure of the helix backbone

Conclusions

Acknowledgments

Biophys. J.

Biophys. J.

J. Mol. Biol.

J. Mol. Biol.

Biophys. J.

Biophys. J.

Trends Biochem. Sci.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Trends Pharmacol. Sci.

Biophys. J.

J. Mol. Biol.

J. Mol. Biol.

Modeling charged protein side chains in lipid membranes

J. Gen. Physiol.

Reconstruction of protein backbones from the brix collection of canonical protein fragments

PLoS Comput. Biol.

Helanal: a program to characterize helix geometry in proteins

J. Biomol. Struct. Dyn.

The Protein Data Bank

Nucleic Acids Res.

Solving the membrane protein folding problem

Nature

AMBER 9