Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.

[1]  O. Hankinson,et al.  Identification of a Novel Domain in the Aryl Hydrocarbon Receptor Required for DNA Binding (*) , 1996, The Journal of Biological Chemistry.

[2]  Annalisa Bordogna,et al.  New Aryl Hydrocarbon Receptor Homology Model Targeted To Improve Docking Reliability , 2011, J. Chem. Inf. Model..

[3]  O. Hankinson,et al.  Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT) , 1994, Molecular and cellular biology.

[4]  Junmei Wang,et al.  Development and testing of a general amber force field , 2004, J. Comput. Chem..

[5]  L. Poellinger,et al.  Protein‐protein interaction via PAS domains: role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor‐Arnt transcription factor complex. , 1995, The EMBO journal.

[6]  H. Swanson,et al.  Cloning and expression of a human Ah receptor cDNA. , 1993, Molecular pharmacology.

[7]  Christopher I. Bayly,et al.  Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: II. Parameterization and validation , 2002, J. Comput. Chem..

[8]  Kevin H. Gardner,et al.  Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor , 2009, Proceedings of the National Academy of Sciences.

[9]  P. Beroza,et al.  Application of a pairwise generalized Born model to proteins and nucleic acids: inclusion of salt effects , 1999 .

[10]  Giorgio Colombo,et al.  Investigating allostery in molecular recognition: insights from a computational study of multiple antibody-antigen complexes. , 2013, The journal of physical chemistry. B.

[11]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[12]  T. Clackson,et al.  A hot spot of binding energy in a hormone-receptor interface , 1995, Science.

[13]  A. Sali,et al.  Statistical potential for assessment and prediction of protein structures , 2006, Protein science : a publication of the Protein Society.

[14]  L. Poellinger,et al.  Definition of a Dioxin Receptor Mutant That Is a Constitutive Activator of Transcription , 2001, The Journal of Biological Chemistry.

[15]  D. Baker,et al.  A simple physical model for binding energy hot spots in protein–protein complexes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Emil Alexov,et al.  Rapid grid‐based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: Applications to the molecular systems and geometric objects , 2002, J. Comput. Chem..

[17]  Daniel J. Peet,et al.  bHLH–PAS proteins in cancer , 2013, Nature Reviews Cancer.

[18]  Manfred J. Sippl,et al.  Thirty years of environmental health research--and growing. , 1996, Nucleic Acids Res..

[19]  R. Russell,et al.  The relationship between sequence and interaction divergence in proteins. , 2003, Journal of molecular biology.

[20]  Ricardo A Broglia,et al.  Understanding the determinants of stability and folding of small globular proteins from their energetics , 2003, Protein science : a publication of the Protein Society.

[21]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[22]  A. Chapman-Smith,et al.  The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. , 2004, The international journal of biochemistry & cell biology.

[23]  Alessandro Pandini,et al.  Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis. , 2009, Biochemistry.

[24]  D. Case,et al.  Exploring protein native states and large‐scale conformational changes with a modified generalized born model , 2004, Proteins.

[25]  Hui Lu,et al.  Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles , 2013, Journal of chemical theory and computation.

[26]  W. C. Still,et al.  Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO) , 1999 .

[27]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[28]  M. Denison,et al.  Role of the Per/Arnt/Sim Domains in Ligand-dependent Transformation of the Aryl Hydrocarbon Receptor* , 2008, Journal of Biological Chemistry.

[29]  Valerie Daggett,et al.  Principles of ligand binding within a completely buried cavity in HIF2alpha PAS-B. , 2009, Journal of the American Chemical Society.

[30]  M. Sippl.  Recognition of errors in three‐dimensional structures of proteins , 1993, Proteins.

[31]  Gregory D. Hawkins,et al.  Parametrized Models of Aqueous Free Energies of Solvation Based on Pairwise Descreening of Solute Atomic Charges from a Dielectric Medium , 1996 .

[32]  Youngchang Kim,et al.  Structural integration in hypoxia-inducible factors , 2015, Nature.

[33]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[34]  C. A. Andersen,et al.  Continuum secondary structure captures protein flexibility. , 2002, Structure.

[35]  A. Sali,et al.  Modeling of loops in protein structures , 2000, Protein science : a publication of the Protein Society.

[36]  Gary W. Caldwell,et al.  Optimization in Drug Discovery , 2014, Methods in Pharmacology and Toxicology.

[37]  Alessandro Pandini,et al.  Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. , 2007, Biochemistry.

[38]  Özkan Yildiz,et al.  Structural and Functional Analyses of PAS Domain Interactions of the Clock Proteins Drosophila PERIOD and Mouse PERIOD2 , 2009, PLoS biology.

[39]  Julie C. Mitchell,et al.  KFC2: A knowledge‐based hot spot prediction method based on interface solvation, atomic density, and plasticity features , 2011, Proteins.

[40]  Achim Kramer,et al.  Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function , 2012, Proceedings of the National Academy of Sciences.

[41]  Holger Gohlke,et al.  MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. , 2012, Journal of chemical theory and computation.

[42]  Thomas H Scheuermann,et al.  Development of inhibitors of the PAS-B domain of the HIF-2α transcription factor. , 2013, Journal of medicinal chemistry.

[43]  Rui Chen,et al.  Allosteric Inhibition of Hypoxia Inducible Factor-2 with Small Molecules , 2013, Nature chemical biology.

[44]  K. Gardner,et al.  Structural basis for PAS domain heterodimerization in the basic helix–loop–helix-PAS transcription factor hypoxia-inducible factor , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Anatoly A. Soshilov,et al.  Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis , 2014, Molecular and Cellular Biology.

[46]  H. Gohlke,et al.  Free Energy Calculations by the Molecular Mechanics Poisson−Boltzmann Surface Area Method , 2012, Molecular informatics.

[47]  Ian B. Dodd,et al.  Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR , 2011, Nucleic acids research.

[48]  O. Hankinson,et al.  Identification of Functional Domains of the Aryl Hydrocarbon Receptor (*) , 1995, The Journal of Biological Chemistry.

[49]  F. Whelan,et al.  The pleiotropy of dioxin toxicity--xenobiotic misappropriation of the aryl hydrocarbon receptor's alternative physiological roles. , 2009, Pharmacology & therapeutics.

[50]  Rainer Breitling,et al.  Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments , 2004, FEBS letters.

[51]  A. Bogan,et al.  Anatomy of hot spots in protein interfaces. , 1998, Journal of molecular biology.

[52]  Hong Zhang,et al.  Crystal Structure of the Heterodimeric CLOCK:BMAL1 Transcriptional Activator Complex , 2012, Science.

[53]  Daniel W. A. Buchan,et al.  Scalable web services for the PSIPRED Protein Analysis Workbench , 2013, Nucleic Acids Res..

[54]  V. Hornak,et al.  Comparison of multiple Amber force fields and development of improved protein backbone parameters , 2006, Proteins.

[55]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[56]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[57]  Youngchang Kim,et al.  Structure and Dimerization Properties of the Aryl Hydrocarbon Receptor PAS-A Domain , 2013, Molecular and Cellular Biology.

[58]  Ozlem Keskin,et al.  HotPoint: hot spot prediction server for protein interfaces , 2010, Nucleic Acids Res..

[59]  Roman A. Laskowski,et al.  LigPlot+: Multiple Ligand-Protein Interaction Diagrams for Drug Discovery , 2011, J. Chem. Inf. Model..

[60]  Bin Zhao,et al.  Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.

[61]  Anatoly A. Soshilov,et al.  DNA Binding (Gel Retardation Assay) Analysis for Identification of Aryl Hydrocarbon (Ah) Receptor Agonists and Antagonists , 2014 .