A High-Throughput Mutational Scan of an Intrinsically Disordered Acidic Transcriptional Activation Domain.
Abstract
Transcriptional activation domains are essential for gene regulation, but their intrinsic disorder and low primary sequence conservation have made it difficult to identify the amino acid composition features that underlie their activity. Here, we describe a rational mutagenesis scheme that deconvolves the function of four activation domain sequence features-acidity, hydrophobicity, intrinsic disorder, and short linear motifs-by quantifying the activity of thousands of variants in vivo and simulating their conformational ensembles using an all-atom Monte Carlo approach. Our results with a canonical activation domain from the Saccharomyces cerevisiae transcription factor Gcn4 reconcile existing observations into a unified model of its function: the intrinsic disorder and acidic residues keep two hydrophobic motifs from driving collapse. Instead, the most-active variants keep their aromatic residues exposed to the solvent. Our results illustrate how the function of intrinsically disordered proteins can be revealed by high-throughput rational mutagenesis.
Submission Details
ID: qv26vTuE4
Submitter: Max Staller
Submission Date: July 10, 2018, 8:25 a.m.
Version: 1
Publication Details
Staller MV;Holehouse AS;Swain-Lenz D;Das RK;Pappu RV;Cohen BA,Cell Syst (2018) A High-Throughput Mutational Scan of an Intrinsically Disordered Acidic Transcriptional Activation Domain. PMID:29525204Additional Information
Study Summary
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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Data Distribution
Studies with similar sequences (approximate matches)
Correlation with other assays (exact sequence matches)
Relevant PDB Entries
| Structure ID | Release Date | Resolution | Structure Title |
|---|---|---|---|
| 1ZTA | 1993-04-15 | THE SOLUTION STRUCTURE OF A LEUCINE-ZIPPER MOTIF PEPTIDE | |
| 1IHQ | 2001-10-03 | GLYTM1BZIP: A CHIMERIC PEPTIDE MODEL OF THE N-TERMINUS OF A RAT SHORT ALPHA TROPOMYOSIN WITH THE N-TERMINUS ENCODED BY EXON 1B | |
| 2OVN | 2007-04-03 | NMR structure of the GCN4 trigger peptide | |
| 1FMH | 2000-11-01 | NMR SOLUTION STRUCTURE OF A DESIGNED HETERODIMERIC LEUCINE ZIPPER | |
| 2LPB | 2012-02-22 | Structure of the complex of the central activation domain of Gcn4 bound to the mediator co-activator domain 1 of Gal11/med15 | |
| 2K8X | 2009-04-28 | GlyTM1b(1-19)zip: A Chimeric Peptide Model of the N-Terminus of a Rat Short Alpha-Tropomyosin with the N-Terminus Encoded by Exon 1b in Complex with TM9d(252-284), a Peptide Model Containing the C Terminus of Alpha-Tropomyosin Encoded by Exon 9d | |
| 5IEW | 2017-03-15 | NMR Structures Show Unwinding of the GCN4p Coiled Coil Superhelix Accompanying Disruption of Ion Pairs at Acidic pH | |
| 2N9B | 2016-06-15 | Solution NMR Structure of Antiparallel Myosin-10:GCN4 Tandem Coiled-Coil | |
| 5IIR | 2017-03-15 | NMR Structures Show Unwinding of the GCN4p Coiled Coil Superhelix Accompanying Disruption of Ion Pairs at Acidic pH | |
| 2G9J | 2006-11-07 | Complex of TM1a(1-14)Zip with TM9a(251-284): a model for the polymerization domain ('overlap region') of tropomyosin, Northeast Structural Genomics Target OR9 | |
| 5IIV | 2017-03-15 | GCN4p pH 1.5 | |
| 1TMZ | 1998-06-17 | TMZIP: A CHIMERIC PEPTIDE MODEL OF THE N-TERMINUS OF ALPHA TROPOMYOSIN, NMR, 15 STRUCTURES | |
| 3AZD | 2011-10-19 | 0.98 | Crystal structure of tropomyosin N-terminal fragment at 0.98A resolution |
| 2WQ1 | 2009-11-03 | 1.08 | GCN4 leucine zipper mutant with three IxxNTxx motifs coordinating bromide |
| 2WQ0 | 2009-11-03 | 1.12 | GCN4 leucine zipper mutant with three IxxNTxx motifs coordinating chloride |
| 4OWI | 2014-05-21 | 1.2 | peptide structure |
| 2WQ3 | 2009-11-03 | 1.22 | GCN4 leucine zipper mutant with three IxxNTxx motifs coordinating chloride and nitrate |
| 2HY6 | 2006-10-24 | 1.25 | A seven-helix coiled coil |
| 2WPZ | 2009-11-03 | 1.25 | GCN4 leucine zipper mutant with two VxxNxxx motifs coordinating chloride |
| 2YNY | 2012-12-12 | 1.35 | Salmonella enterica SadA 255-302 fused to GCN4 adaptors (SadAK1) |
| 2IPZ | 2007-01-16 | 1.35 | A Parallel Coiled-Coil Tetramer with Offset Helices |
| 5APU | 2016-01-27 | 1.35 | Sequence IANKEDKAD inserted between GCN4 adaptors - Structure A9b black |
| 2WQ2 | 2009-11-03 | 1.36 | GCN4 leucine zipper mutant with three IxxNTxx motifs coordinating iodide |
| 5APS | 2016-01-27 | 1.37 | Sequence IENKKAD inserted between GCN4 adaptors - Structure A7 |
| 2NRN | 2007-04-17 | 1.4 | Self-assembly of coiled-coil tetramers in the 1.40 A structure of a leucine-zipper mutant |
| 2YNZ | 2012-12-12 | 1.4 | Salmonella enterica SadA 823-947 fused to a GCN4 adaptor (SadAK5) |
| 3M48 | 2011-02-16 | 1.45 | GCN4 Leucine Zipper Peptide Mutant |
| 5KHT | 2017-06-21 | 1.5 | Crystal structure of the N-terminal fragment of tropomyosin isoform Tpm1.1 at 1.5 A resolution |
| 1LLM | 2003-09-30 | 1.5 | Crystal Structure of a Zif23-GCN4 Chimera Bound to DNA |
| 2BNI | 2005-03-30 | 1.5 | pLI mutant E20C L16G Y17H, antiparallel |
| 2B1F | 2006-01-31 | 1.5 | Antiparallel four-stranded coiled coil specified by a 3-3-1 hydrophobic heptad repeat |
| 5APZ | 2016-01-27 | 1.6 | Thermosinus carboxydivorans Nor1 Tcar0761 residues 68-101 and 191-211 fused to GCN4 adaptors |
| 2CCN | 2006-04-06 | 1.6 | pLI E20C is antiparallel |
| 5APW | 2016-01-27 | 1.6 | Sequence MATKDD inserted between GCN4 adaptors - Structure T6 |
| 3I1G | 2009-07-07 | 1.6 | Crystal structure of a GCN4 leucine zipper mutant at 1.6 A resolution |
| 3CRP | 2008-10-07 | 1.7 | A heterospecific leucine zipper tetramer |
| 1UO4 | 2004-10-13 | 1.7 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 2CCF | 2006-04-06 | 1.7 | Antiparallel Configuration of pLI E20S |
| 1IJ2 | 2001-08-08 | 1.7 | GCN4-pVTL Coiled-coil Trimer with Threonine at the a(16) position |
| 3CK4 | 2008-10-07 | 1.7 | A heterospecific leucine zipper tetramer |
| 2WPY | 2009-11-03 | 1.75 | GCN4 leucine zipper mutant with one VxxNxxx motif coordinating chloride |
| 5APT | 2016-01-27 | 1.8 | Sequence IENKADKAD inserted between GCN4 adaptors - Structure A9 |
| 2VNL | 2009-02-10 | 1.8 | MUTANT Y108Wdel OF THE HEADBINDING DOMAIN OF PHAGE P22 TAILSPIKE C- TERMINally fused to ISOLEUCINE ZIPPER pIIGCN4 (chimera II) |
| 5APX | 2016-01-27 | 1.8 | Sequence MATKDDIAN inserted between GCN4 adaptors - Structure T9(6) |
| 2EFR | 2008-03-04 | 1.8 | Crystal structure of the c-terminal tropomyosin fragment with N- and C-terminal extensions of the leucine zipper at 1.8 angstroms resolution |
| 1GCM | 1996-01-29 | 1.8 | GCN4 LEUCINE ZIPPER CORE MUTANT P-LI |
| 1PIQ | 1998-09-30 | 1.8 | CRYSTAL STRUCTURE OF GCN4-PIQ, A TRIMERIC COILED COIL WITH BURIED POLAR RESIDUES |
| 6O2F | 2019-06-26 | 1.8 | GCN4 with NPEG4 at position 18 |
| 1CE9 | 1999-03-25 | 1.8 | HELIX CAPPING IN THE GCN4 LEUCINE ZIPPER |
| 2ZTA | 1992-10-15 | 1.8 | X-RAY STRUCTURE OF THE GCN4 LEUCINE ZIPPER, A TWO-STRANDED, PARALLEL COILED COIL |
| 1IJ3 | 2001-08-08 | 1.8 | GCN4-pVSL Coiled-coil trimer with Serine at the a(16) position |
| 4TL1 | 2014-08-06 | 1.8 | GCN4-p1 with mutation to 1-Aminocyclohexanecarboxylic acid at residue 10 |
| 1ZIK | 1997-07-07 | 1.8 | GCN4-LEUCINE ZIPPER CORE MUTANT ASN16LYS IN THE DIMERIC STATE |
| 1ZII | 1997-07-07 | 1.8 | GCN4-LEUCINE ZIPPER CORE MUTANT ASN16ABA IN THE DIMERIC STATE |
| 1GZL | 2002-10-10 | 1.8 | Crystal structure of C14linkmid/IQN17: a cross-linked inhibitor of HIV-1 entry bound to the gp41 hydrophobic pocket |
| 3ZMF | 2013-02-20 | 1.85 | Salmonella enterica SadA 303-358 fused to GCN4 adaptors (SadAK2) |
| 1IJ0 | 2001-08-08 | 1.86 | Coiled Coil Trimer GCN4-pVLS Ser at Buried D Position |
| 1IJ1 | 2001-08-08 | 1.86 | GCN4-pVLT Coiled-coil Trimer with Threonine at the d(12) Position |
| 2O7H | 2007-11-27 | 1.86 | Crystal structure of trimeric coiled coil GCN4 leucine zipper |
| 4G2K | 2012-09-12 | 1.9 | Crystal structure of the Marburg Virus GP2 ectodomain in its post-fusion conformation |
| 1GK6 | 2002-03-15 | 1.9 | Human vimentin coil 2B fragment linked to GCN4 leucine zipper (Z2B) |
| 3K7Z | 2009-11-24 | 1.9 | GCN4-Leucine zipper core mutant as N16A trigonal automatic solution |
| 1RB5 | 2004-01-13 | 1.9 | ANTIPARALLEL TRIMER OF GCN4-LEUCINE ZIPPER CORE MUTANT AS N16A TRIGONAL FORM |
| 1RB4 | 2004-01-13 | 1.9 | ANTIPARALLEL TRIMER OF GCN4-LEUCINE ZIPPER CORE MUTANT AS N16A TETRAGONAL AUTOMATIC SOLUTION |
| 6O2E | 2019-06-26 | 1.9 | GCN4 with asparagine at position 18 |
| 2CCE | 2006-04-06 | 1.9 | Parallel Configuration of pLI E20S |
| 4NJ0 | 2014-08-20 | 1.9 | GCN4-p1 single Val9 to Ile mutant |
| 1RB6 | 2004-01-13 | 1.9 | ANTIPARALLEL TRIMER OF GCN4-LEUCINE ZIPPER CORE MUTANT AS N16A TETRAGONAL FORM |
| 1UO3 | 2004-10-13 | 1.92 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1W5K | 2004-09-24 | 1.92 | AN ANTI-PARALLEL FOUR HELIX BUNDLE |
| 6E52 | 2019-02-27 | 1.93 | Chimeric structure of Saccharomyces cerevisiae GCN4 leucine zipper fused to Staphylococcus aureus AgrC cytoplasmic histidine kinase module (dataset anisotropically truncated by STARANISO) |
| 3I5C | 2009-08-18 | 1.94 | Crystal structure of a fusion protein containing the leucine zipper of GCN4 and the GGDEF domain of WspR from Pseudomonas aeruginosa |
| 4C46 | 2013-09-11 | 1.95 | ANDREI-N-LVPAS fused to GCN4 adaptors |
| 1UO2 | 2004-10-13 | 1.99 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1LD4 | 2002-11-04 | 11.4 | Placement of the Structural Proteins in Sindbis Virus |
| 4NJ1 | 2014-08-20 | 2.0 | GCN4-p1 double Val9, 23 to Ile mutant |
| 4NIZ | 2014-08-20 | 2.0 | GCN4-p1 single Val9 to aminobutyric acid mutant |
| 5APV | 2016-01-27 | 2.0 | Sequence IANKEDKAD inserted between GCN4 adaptors - Structure A9b grey |
| 1ZIJ | 1997-07-07 | 2.0 | GCN4-LEUCINE ZIPPER CORE MUTANT ASN16ABA IN THE TRIMERIC STATE |
| 2AHP | 2006-08-01 | 2.0 | GCN4 leucine zipper, mutation of Lys15 to epsilon-azido-Lys |
| 5APY | 2016-01-27 | 2.0 | Sequence MATKDDIAN inserted between GCN4 adaptors - Structure T9(9) |
| 2B22 | 2006-01-31 | 2.0 | Antiparallel four-stranded coiled coil specified by a 3-3-1 hydrophobic heptad repeat |
| 3G9R | 2009-05-12 | 2.0 | Structure of the HIV-1 gp41 Membrane-Proximal Ectodomain Region in a Putative Prefusion Conformation |
| 2EFS | 2008-03-04 | 2.0 | Crystal structure of the C-terminal tropomyosin fragment with N- and C-terminal extensions of the leucine zipper at 2.0 angstroms resolution |
| 2YO3 | 2012-12-12 | 2.0 | Salmonella enterica SadA 1185-1386 fused to GCN4 adaptors (SadAK14) |
| 2YO2 | 2012-12-12 | 2.0 | Salmonella enterica SadA 255-358 fused to GCN4 adaptors (SadAK12) |
| 1ZIM | 1997-07-07 | 2.0 | GCN4-LEUCINE ZIPPER CORE MUTANT ASN16GLN IN THE TRIMERIC STATE |
| 3GJP | 2010-03-09 | 2.0 | Crystal structure of mutant coiled coil GCN4 leucine zipper |
| 4DMD | 2012-11-14 | 2.0 | GCN4 leucine zipper domain in a dimeric oligomerization state |
| 2VKY | 2009-02-10 | 2.05 | Headbinding Domain of Phage P22 Tailspike C-Terminally Fused to Isoleucine Zipper pIIGCN4 (Chimera I) |
| 1UNU | 2004-10-13 | 2.07 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1UO5 | 2004-10-13 | 2.07 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1UNT | 2004-10-13 | 2.07 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 5APQ | 2016-01-27 | 2.1 | Sequence IENKAD inserted between GCN4 adaptors - Structure A6 |
| 6H9M | 2019-02-13 | 2.1 | Coiled-coil domain-containing protein 90B residues 43-125 from Homo sapiens fused to a GCN4 adaptor |
| 1GCL | 1995-06-03 | 2.1 | GCN4 LEUCINE ZIPPER CORE MUTANT P-LI |
| 2WG5 | 2009-04-28 | 2.1 | Proteasome-Activating Nucleotidase (PAN) N-domain (57-134) from Archaeoglobus fulgidus fused to GCN4 |
| 2Z5I | 2008-04-22 | 2.1 | Crystal structure of the head-to-tail junction of tropomyosin |
| 1UNV | 2004-10-13 | 2.14 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1W5G | 2004-09-24 | 2.16 | An anti-parallel four helix bundle (acetimide modification). |
| 1W5L | 2004-09-24 | 2.17 | An anti-parallel to parallel switch. |
| 1W5J | 2004-09-24 | 2.2 | AN ANTI-PARALLEL FOUR HELIX BUNDLE |
| 1UNW | 2004-10-13 | 2.2 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 2DGC | 1996-03-08 | 2.2 | GCN4 BASIC DOMAIN, LEUCINE ZIPPER COMPLEXED WITH ATF/CREB SITE DNA |
| 4NJ2 | 2014-08-20 | 2.2 | GCN4-p1 triple Val9, 23,30 to Ile mutant |
| 1ZIL | 1997-07-07 | 2.25 | GCN4-LEUCINE ZIPPER CORE MUTANT ASN16GLN IN THE DIMERIC STATE |
| 6E95 | 2019-02-27 | 2.25 | Chimeric structure of Saccharomyces cerevisiae GCN4 leucine zipper fused to Staphylococcus aureus AgrC cytoplasmic histidine kinase module (dataset isotropically truncated by HKL2000) |
| 4HU6 | 2013-08-21 | 2.3 | Oxime side-chain cross-links in the GCN4-p1 dimeric coiled coil: Cyclic product |
| 3BAS | 2008-01-08 | 2.3 | Crystal structure of the N-terminal region of the scallop myosin rod, monoclinic (C2) form |
| 4HU5 | 2013-08-21 | 2.3 | Oxime side-chain cross-links in the GCN4-p1 dimeric coiled coil: Linear precursor |
| 5APP | 2016-01-27 | 2.3 | Actinobacillus actinomycetemcomitans OMP100 residues 133-198 fused to GCN4 adaptors |
| 1W5I | 2004-09-24 | 2.3 | ABA does not affect topology of pLI. |
| 1UNZ | 2004-10-13 | 2.3 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 3BAT | 2008-01-08 | 2.3 | Crystal structure of the N-terminal region of the scallop myosin rod, monoclinic (P21) form |
| 1UNY | 2004-10-13 | 2.3 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1UO0 | 2004-10-13 | 2.4 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1UNX | 2004-10-13 | 2.4 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 2WG6 | 2009-04-28 | 2.5 | Proteasome-Activating Nucleotidase (PAN) N-domain (57-134) from Archaeoglobus fulgidus fused to GCN4, P61A Mutant |
| 1NKN | 2003-07-22 | 2.5 | VISUALIZING AN UNSTABLE COILED COIL: THE CRYSTAL STRUCTURE OF AN N-TERMINAL SEGMENT OF THE SCALLOP MYOSIN ROD |
| 1UO1 | 2004-10-13 | 2.5 | Structure Based Engineering of Internal Molecular Surfaces Of Four Helix Bundles |
| 1W5H | 2004-09-24 | 2.5 | An anti-parallel four helix bundle. |
| 1SWI | 1996-11-08 | 2.6 | GCN4-LEUCINE ZIPPER CORE MUTANT AS N16A COMPLEXED WITH BENZENE |
| 2D3E | 2006-09-12 | 2.6 | Crystal structure of the C-Terminal fragment of rabbit skeletal alpha-tropomyosin |
| 1KQL | 2002-05-29 | 2.7 | Crystal structure of the C-terminal region of striated muscle alpha-tropomyosin at 2.7 angstrom resolution |
| 2YO0 | 2012-12-12 | 2.8 | Salmonella enterica SadA 1049-1304 fused to GCN4 adaptors (SadAK9-cfI) |
| 2Z5H | 2008-04-22 | 2.89 | Crystal structure of the head-to-tail junction of tropomyosin complexed with a fragment of TnT |
| 3P8M | 2011-08-31 | 2.9 | Human dynein light chain (DYNLL2) in complex with an in vitro evolved peptide dimerized by leucine zipper |
| 1YSA | 1993-10-31 | 2.9 | THE GCN4 BASIC REGION LEUCINE ZIPPER BINDS DNA AS A DIMER OF UNINTERRUPTED ALPHA HELICES: CRYSTAL STRUCTURE OF THE PROTEIN-DNA COMPLEX |
| 1FAV | 2000-08-23 | 3.0 | THE STRUCTURE OF AN HIV-1 SPECIFIC CELL ENTRY INHIBITOR IN COMPLEX WITH THE HIV-1 GP41 TRIMERIC CORE |
| 1DGC | 1994-06-22 | 3.0 | THE X-RAY STRUCTURE OF THE GCN4-BZIP BOUND TO ATF/CREB SITE DNA SHOWS THE COMPLEX DEPENDS ON DNA FLEXIBILITY |
| 2YO1 | 2012-12-12 | 3.1 | Salmonella enterica SadA 1049-1304 fused to GCN4 adaptors (SadAK9- cfII) |
| 6HN5 | 2018-11-21 | 3.2 | Leucine-zippered human insulin receptor ectodomain with single bound insulin - 'upper' membrane-distal part |
| 6HN4 | 2018-11-21 | 4.2 | Leucine-zippered human insulin receptor ectodomain with single bound insulin - 'lower' membrane-proximal part |
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 100.0 | General control protein GCN4 -Central acidic activation domain (101-144) | P03069 | GCN4_YEAST |