Coupling of ligand binding and dimerization of helix-loop-helix peptides: Spectroscopic and sedimentation analyses of calbindin D9k EF-hands

Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca2+-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca2+-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pKa of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca2+-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg2+-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca2+/Mg2+ affinity of the EF site, and accelerates Ca2+ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca2+ affinity.

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Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8343-8355.1994 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8343-8355

Author(s):

M L Whitelaw ◽

J A Gustafsson ◽

L Poellinger

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Ligand Binding ◽

Transactivation Domain ◽

Response Element ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

C Terminus ◽

Heterodimeric Complex ◽

Dioxin Receptor

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

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Structures and metal-ion-binding properties of the Ca2+-binding helix–loop–helix EF-hand motifs

Biochemical Journal ◽

10.1042/bj20070255 ◽

2007 ◽

Vol 405 (2) ◽

pp. 199-221 ◽

Cited By ~ 525

Author(s):

Jessica L. Gifford ◽

Michael P. Walsh ◽

Hans J. Vogel

Keyword(s):

Metal Ion ◽

Molten Globule ◽

Family Members ◽

Binding Motif ◽

Metal Ion Binding ◽

Protein Target ◽

Helix Loop Helix ◽

Wide Range ◽

Ef Hand ◽

Ef Hands

The ‘EF-hand’ Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix–loop–helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the ‘canonical’) EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca2+ signal required to reach protein saturation. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca2+: selectivity over Mg2+ (a cation with very similar chemical properties to Ca2+ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.

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Symmetrical Stabilization of Bound Ca2+Ions in a Cooperative Pair of EF-Hands through Hydrogen Bonding of Coordinating Water Molecules in Calbindin D9k†

Biochemistry ◽

10.1021/bi010551h ◽

2001 ◽

Vol 40 (33) ◽

pp. 9887-9895 ◽

Cited By ~ 6

Author(s):

Jonas Fast ◽

Maria Håkansson ◽

Andreas Muranyi ◽

Garry P. Gippert ◽

Eva Thulin ◽

...

Keyword(s):

Hydrogen Bonding ◽

Water Molecules ◽

Calbindin D9k ◽

Ef Hands

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NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

eLife ◽

10.7554/elife.18790 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 22

Author(s):

Dalei Wu ◽

Xiaoyu Su ◽

Nalini Potluri ◽

Youngchang Kim ◽

Fraydoon Rastinejad

Keyword(s):

Transcription Factors ◽

Ligand Binding ◽

Crystal Structures ◽

Autism Spectrum ◽

Helix Loop Helix ◽

Aryl Hydrocarbon ◽

Binding Pockets ◽

Direct Targeting ◽

Spectrum Disorders ◽

Transcription Complexes

The neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the wider mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.

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Homology modelling of integrin EF-hands. Evidence for widespread use of a conserved cation-binding site

Biochemical Journal ◽

10.1042/bj2850325 ◽

1992 ◽

Vol 285 (1) ◽

pp. 325-331 ◽

Cited By ~ 86

Author(s):

D S Tuckwell ◽

A Brass ◽

M J Humphries

Keyword(s):

Ligand Binding ◽

Homology Modelling ◽

Cation Binding ◽

Binding Motif ◽

Binding Model ◽

Bivalent Cation ◽

Cation Binding Site ◽

Ef Hand ◽

Ef Hands

Integrin alpha-subunits contain three or four peptide sequences that are similar to the EF-hand, a 13-residue bivalent cation-binding motif found in calmodulin and parvalbumin. The integrin sequences differ from classical EF-hands in that they lack a co-ordinating residue at position 12. One hypothesis to explain integrin-ligand binding is that aspartate-containing recognition sequences in integrin ligands, which bind at or near to the EF-hand-like sequences, may take the place of the missing residue and co-ordinate directly to the bound cation. In this report, homology modelling of integrin EF-hand-like sequences has been performed using the X-ray structure of calmodulin as a template in order to assess the functional activity of the integrin sequences. In the calmodulin-integrin hybrid structures, integrin EF-hand-like sequences were able to retain cations whereas control sequences did not. Structural analyses demonstrated that the integrin sequences in the hybrid proteins closely resembled conventional EF-hands. The integrin sequences are therefore highly likely to bind Ca2+ ions in vivo, a prerequisite for the ligand-binding model. Database searching with a matrix derived from known integrin EF-hand-like sequences has been used to identify other proteins containing the integrin EF-hand-like motif. Annexin V (anchorin CII), atrial natriuretic peptide receptors and the 70 kDa heat-shock protein were identified by the matrix; the functions of these proteins are known from previous studies to be bivalent cation-dependent. These findings suggest that the integrin EF-hand-like sequence may be a more common motif than originally thought.

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Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8343 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8343-8355 ◽

Cited By ~ 120

Author(s):

M L Whitelaw ◽

J A Gustafsson ◽

L Poellinger

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Ligand Binding ◽

Transactivation Domain ◽

Response Element ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

C Terminus ◽

Heterodimeric Complex ◽

Dioxin Receptor

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

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Focusing of the electrostatic potential at EF-hands of calbindin D9k: Titration of acidic residues

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.1132 ◽

2001 ◽

Vol 45 (2) ◽

pp. 129-135 ◽

Cited By ~ 20

Author(s):

Tõnu Kesvatera ◽

Bo Jönsson ◽

Eva Thulin ◽

Sara Linse

Keyword(s):

Electrostatic Potential ◽

Calbindin D9k ◽

Acidic Residues ◽

Ef Hands

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Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.756 ◽

1995 ◽

Vol 15 (2) ◽

pp. 756-765 ◽

Cited By ~ 80

Author(s):

C Antonsson ◽

M L Whitelaw ◽

J McGuire ◽

J A Gustafsson ◽

L Poellinger

Keyword(s):

Dna Binding ◽

Ligand Binding ◽

Molecular Chaperone ◽

Wheat Germ ◽

Binding Activity ◽

Pas Domain ◽

Receptor Function ◽

Helix Loop Helix ◽

Reticulocyte Lysate ◽

Dioxin Receptor

The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.

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Biophysical Approaches Determining Ligand Binding to Biomolecular Targets

10.1039/9781849732666 ◽

2011 ◽

Cited By ~ 6

Keyword(s):

Ligand Binding

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