Long-Range Coupling in an Allosteric Receptor Revealed by Mutant Cycle Analysis

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50–60 Å from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (βL9′S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC50 measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors

Evidence for an Extended Hydrogen Bond Network in the Binding Site of the Nicotinic Receptor: Role of the Vicinal Disulfide of the α1 Subunit

The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a β turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the β turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp

Chemical Scale Studies of the Phe-Pro Conserved Motif in the Cys Loop of Cys

The functions of two conserved residues, Phe^(135) and Pro^(136), located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe^(135), backbone conformation at Pro^(136), side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function

Chemical-Scale Studies of the Nicotinic Acetylcholine Receptor: Insights from Amide-to-Ester Backbone Mutagenesis

This thesis describes the use of peptide backbone amide-to-ester mutations to study the structure and function of ligand-gated ion channels. The research described herein has been done on the muscle nicotinic acetylcholine receptor, a prototypical ligand-gated ion channel in the cys-loop superfamily. Backbone mutagenesis in these proteins provides insight into specific intermolecular interactions that are critical to function, as well as answering more fundamental questions about the role of the peptide backbone in long-range conformational changes in these allosteric receptors. Chapter 2 describes the identification of a key hydrogen bond near the binding site that is involved in the gating pathway. We found that the backbone N-H of a loop C residue makes a hydrogen bond to an anionic side chain of the complementary subunit upon agonist binding. The hydrogen bonding partner is not the residue predicted by structural data, but instead an aspartate that was originally believed to participate directly in agonist binding. In chapter 3 we consider the involvement of the peptide backbone in the binding-induced conformational changes that lead to channel gating. Single backbone mutations in the β-sheet-rich extracellular domain were well tolerated, whereas two proximal backbone mutations led to nonfunctional receptors. These results support a model in which backbone movements in the outer β-sheet are important for receptor function. Chapter 4 describes a new method - elucidating long-range functional coupling in allosteric receptors (ELFCAR) - that should be broadly applicable to determining functional roles of residues in allosteric receptors. Chapters 5 and 6 describe electrophysiological and computational investigations into the role of amide-to-ester mutations in the aromatic binding box of the nicotinic receptor. Echoing the results of chapter 3, these mutations largely reveal an overall tolerance of backbone mutations in the binding site. Finally, in chapter 7, we explore the use of ester and N-methyl backbone modifications to uncover the role of conformational changes at an unusual vicinal disulfide bond near the tip of the C-loop. Using ab initio calculations, we demonstrate that N-methylation and esterification of this ring structure in model peptides dramatically impacts its cis-trans conformational preferences.</p

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH_2-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH_2-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements