Aromatic interactions in homeodomains contribute to the low quantum yield of a conserved, buried tryptophan

Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart Na V 1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the Na V 1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na ؉ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it.Channelopathies, so named because they represent a set of diseases caused by mutations in genes coding for ion channels, are a new and growing class of human disorders that include but are not limited to diabetes, muscle disorders, neurological disease, and cardiac arrhythmias (1). A surprising number of channelopathies associated with a wide diversity of human disease are caused by similar mutation-induced changes in ion channel function. Inactivation of voltage-dependent Na ϩ channels is an example of a physiological process critically important in many tissues that, when altered by mutation, can result in muscle weakness, inherited epilepsies, autism, or cardiac arrhythmia (2-4). Clinical consequences of inherited mutations that disrupt Na ϩ channel inactivation provide the most direct link between ion channel biophysics and human physiology and pathophysiology. Conversely, investigation into the consequences of inherited mutations on ion channel function has, in many cases, provided insight into the physiological importance of novel regions and/or structures of ion channel proteins.In the heart, Na ϩ channels (Na V 1.5) 4 primarily underlie action potential initiation and propagation but more recently have been shown to be critical determinants of action potential duration, particularly in the setting of certain inherited channelopathies. Inherited mutations in SCN5A, the gene coding for Na V 1.5, are now known to underlie multiple inherited cardiac arrhythmias, including the congenital long QT syndrome variant 3, Brugada syndrome, and isolated conduction disease (5), and in most cases, these inherited mutations disrupt channel inactivation.Fast inactivation of Na ϩ channels is due to rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization...

Section: Thermodynamic Cycle Analysis Is Consistent With Predictedsupporting

confidence: 64%

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Glaaser

Bankston

Liu³

et al. 2006

“…The interactions may also occur more widely in protein structures: a data base search has found similar indole-water interactions that appear to support certain peptide geometries in other folds. 6 Finally, we note that the homeodomains exhibit a distinctive tryptophan fluorescence quenching (39,40), and we propose that this is caused by the water-indole interaction. Leu 26 Network: Implications for Induced Fit-We have identified a network of residues that mutually pack in several different ways.…”

Section: Fig 3 a Novel Indole-water Interaction In En-hdmentioning

confidence: 69%

“…All data were processed in FELIX 98 (Biosym Technologies, San Diego, CA). 15 N T 1 and T 2 experiments incorporated spin-echo delay times of 500 s, the relaxation times were set to 10,20,40,60,80,120,200,240,280,320, and 360 ms. The recycle delays for the T 2 experiments were 3.0 s. Each spectrum comprised 512 ϫ 128 complex points that were zerofilled 2-fold in both dimensions and multiplied by exponential window functions in both dimensions prior to analysis.…”

Section: Methodsmentioning

confidence: 99%

“…6 The water-indole interaction could explain the distinctive fluorescence properties of homeodomains. With refolding, fluorescence quenching is observed for the En-HD, antennapedia, bicoid, and ubx homeodomains (39,40) as well as in paired, msx-1, oct-1, oct-2, and pit-1 homeodomains. 7 It has been argued that the aromatic residue at position 8 is largely responsible for the quenching as a consequence of its system receiving a H-bond from the indole nitrogen (40).…”

Section: Fig 2 Structure and Electrostatic Surfaces Of En-hd And Itmentioning

confidence: 99%

See 1 more Smart Citation

Crystal Structures of Engrailed Homeodomain Mutants

Stollar¹,

Mayor

Lovell³

et al. 2003

We report the crystal structures and biophysical characterization of two stabilized mutants of the Drosophila Engrailed homeodomain that have been engineered to minimize electrostatic repulsion. Four independent copies of each mutant occupy the crystal lattice, and comparison of these structures illustrates variation that can be partly ascribed to networks of correlated conformational adjustments. Central to one network is leucine 26 (Leu 26 ), which occupies alternatively two side chain rotameric conformations (-gauche and trans) and different positions within the hydrophobic core. Similar sets of conformational substates are observed in other Engrailed structures and in another homeodomain. The pattern of structural adjustments can account for NMR relaxation data and sequence co-variation networks in the wider homeodomain family. It may also explain the dysfunction associated with a P26L mutation in the human ARX homeodomain protein. Finally, we observe a novel dipolar interaction between a conserved tryptophan and a water molecule positioned along the normal to the indole ring. This interaction may explain the distinctive fluorescent properties of the homeodomain family.The homeodomain is a simple fold common to many different DNA-binding proteins from diverse eukaryotes. The domain is found in transcription factors that regulate a variety of genes and so control key processes ranging from early developmental decision to homeostasis and general "housekeeping" (1-3).The homeodomain fold comprises three helices, connected by a flexible loop and a ␤-turn, and has a small hydrophobic core that is remarkably well conserved (4). The side chains in this conserved core appear to be well packed, which is a hallmark of stable folds. However, homeodomain folds generally have poorer stabilities in comparison with other proteins of similar size (5-8). The termini of homeodomains are disordered in solution (9 -11) and structures of free-and DNA-bound forms illustrate that DNA binding is accompanied by a structural condensation of the termini (12). Possibly, the homeodomain core also undergoes subtle structural changes upon DNA binding. Thus, the homeodomain would appear to mold to the surface of specific DNA by induced fit. Despite the wealth of current stereochemical and functional data for homeodomains, the basis for their comparatively low stability and its relationship, if any, to their induced fit remains to be established.Sequence co-variance analysis of homeodomains highlights several strongly correlated residue pairs that form conserved interactions and that may affect stability and DNA binding (4). One pair identified was the surface residues 17 and 52, which forms, in the majority of cases, a salt bridge. However, in the transcriptional repressor Engrailed from Drosophila, two lysines are instead found in these positions. We have substituted lysine 52 for glutamate, to mimic the conserved Glu 17 -Arg 52 salt bridge, and for alanine, to relieve the repulsion of the clustered positive charges. Standard equilibrium ...

“…In homeodomains, Trp-48 exhibits a strongly quenched fluorescence in the native state (22)(23)(24) most likely because of specific interactions with neighboring residues. Conservative mutation of the strictly conserved Trp-48 to a phenylalanine (W48F) reduces DNA binding affinity and destabilizes the HD, suggesting that the role of Trp-48 is to stabilize critical structural features required for DNA recognition.…”

mentioning

confidence: 99%

Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Subramaniam¹,

Jovin

Rivera‐Pomar

2001

The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription and translation factor dependent on the unique DNA and RNA binding properties of the homeodomain (HD). We have used circular dichroism and fluorescence spectroscopy to probe the structure and stability of the Bcd-HD, for which a high resolution structure is not yet available. The fluorescence from the single tryptophan residue in the HD (Trp-48) is strongly quenched in the native state but is dramatically enhanced (ϳ20-fold) upon denaturation. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan fluorescence may be a general phenomenon of HD proteins. We have used site-directed mutagenesis to explore the role of aromatic acids in the structure of the Bcd-HD and to evaluate the proposal that interactions between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native state. We determined that both Trp-48 and Phe-8 in the N-terminal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientation of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.The regulation of gene expression is generally accomplished by interactions of proteins with nucleic acids. These regulators contain DNA binding domains (transcription factors) or RNA binding domains (translational regulators). The homeobox encodes a 60-amino acid nucleic acid binding domain, the homeodomain (HD), 1 which is present in many eukaryotic transcription factors and plays a critical role in development (1, 2). The sequences have been well conserved in the course of evolution from fungi to vertebrates. In addition to binding DNA, thereby activating transcription, the Drosophila Bcd-HD has been recently shown to interact with RNA (3-5). Thus, Bcd has a dual role in development, directing pattern formation along the anterior-posterior axis of the embryo by cooperative DNA binding and subsequent gene activation (6 -8) and by binding the mRNA of the regulatory gene caudal (cad), thereby repressing its translation (3, 5).Because of this unique DNA and RNA binding capability, Bcd-HD is a particularly compelling target for investigations of molecular structure and nucleic acid recognition mechanisms.The HD has also become a paradigm for studying DNAprotein interactions. HDs fold into a module with a characteristic structure comprising three ␣-helices. Structural studies, first elucidated for the Antennapedia and Engrailed HDs, show that the three helices of the HD are stabilized by hydrophobic interactions involving residues Leu-16, Trp-48, and Phe-49, which are conserved in all HDs (9 -15). The HD shows a common helix-turn-helix motif encompassing the recognition helix III, which docks into the major groove of DNA. The specificity of DNA recognition is achieved by specific amino acid residuebase contacts in helix III (16), e.g. amino acid residue 50 (...