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PDBsum entry 2flu

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protein Protein-protein interface(s) links
Ligase PDB id
2flu
Jmol
Contents
Protein chains
285 a.a. *
16 a.a. *
Waters ×333
* Residue conservation analysis
PDB id:
2flu
Name: Ligase
Title: Crystal structure of the kelch-neh2 complex
Structure: Kelch-like ech-associated protein 1. Chain: x. Fragment: kelch domain of human keap1. Engineered: yes. Nrf2. Chain: p. Fragment: 16-mer peptide from huma neh2. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: synthesized peptide
Biol. unit: Dimer (from PQS)
Resolution:
1.50Å     R-factor:   0.180     R-free:   0.199
Authors: X.Li,J.Lo,L.Beamer,M.Hannink
Key ref:
S.C.Lo et al. (2006). Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling. EMBO J, 25, 3605-3617. PubMed id: 16888629 DOI: 10.1038/sj.emboj.7601243
Date:
06-Jan-06     Release date:   15-Aug-06    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q14145  (KEAP1_HUMAN) -  Kelch-like ECH-associated protein 1
Seq:
Struc:
 
Seq:
Struc:
624 a.a.
285 a.a.
Protein chain
Pfam   ArchSchema ?
Q16236  (NF2L2_HUMAN) -  Nuclear factor erythroid 2-related factor 2
Seq:
Struc:
 
Seq:
Struc:
605 a.a.
16 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain

 

 
DOI no: 10.1038/sj.emboj.7601243 EMBO J 25:3605-3617 (2006)
PubMed id: 16888629  
 
 
Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling.
S.C.Lo, X.Li, M.T.Henzl, L.J.Beamer, M.Hannink.
 
  ABSTRACT  
 
Keap1 is a BTB-Kelch substrate adaptor protein that regulates steady-state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of Keap1 bound to a 16-mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel beta-strands connected by two overlapping type I beta-turns stabilized by the aspartate and threonine residues. The beta-turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in Keap1. Mutagenesis experiments confirmed the role of individual amino acids for binding of Nrf2 to Keap1 and for Keap1-mediated repression of Nrf2-dependent gene expression. Our results provide a detailed picture of how a BTB-Kelch substrate adaptor protein binds to its cognate substrate and will enable the rational design of novel chemopreventive agents.
 
  Selected figure(s)  
 
Figure 3.
Figure 3 (A) A ribbon diagram of the Kelch -propeller (red) and bound Nrf2 peptide (yellow tube). The termini of the peptide are labeled N-ter and C-ter; those of the Kelch domain are labeled N and C. The six blades of the -propeller are labeled I–VI and the four -strands found in each blade are labeled A–D (white font) on blade VI. (B) Side view of the Kelch domain, with a surface representation of the -propeller. (C) A surface representation of the Kelch propeller (gray) and peptide (yellow tube). Selected residues are shown in blue (basic), orange (polar), and green (apolar).
Figure 4.
Figure 4 (A) Contacts between the side chain atoms of the Nrf2 peptide and residues in the Kelch domain. (B) Contacts between the backbone atoms of the peptide and residues of the Kelch domain. (C) A stick model of the Nrf2 peptide, with intramolecular hydrogen bonds highlighted. An F[o]-F[c] electron density omit map contoured at 2.5 for the vicinity of the peptide is shown in blue. Phases for the map were determined immediately following molecular replacement, before the inclusion of the peptide in the model. The side chain of E78 was not well ordered in the electron density maps and is shown as semi-transparent. (D) A schematic showing both backbone and side-chain contacts to the Nrf2 peptide (yellow/blue) from interacting residues in the Kelch domain.
 
  The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 3605-3617) copyright 2006.  
  Figures were selected by the author.  
 
 
    Author's comment    
 
  The structure of the Kelch domain of the human Keap1 protein bound to a 16-mer peptide from the human Nrf2 protein containing a highly conserved DxETGE motif, residues 77 to 82, was determined to 1.5 Å. Isothermal titration calorimetry revealed that the 16-mer peptide bound to the Kelch domain of Keap1 with a Kd value of 20 nM. The Nrf2-derived peptide binds in the shallow pocket defined by the D-A and B-C loops on the top face of the Kelch domain and buries 420 Å2 of surface area on the Kelch b-propeller. The peptide has two antiparallel b-strands connected by a turn region that has two overlapping type I b-turns (residues 77 to 80 and 78 to 81). The turn region is stabilized by hydrogen bonds involving the side chains of D77 and T80 and the peptide backbone. Phosphorylation of the highly conserved threonine residue disrupts the b-turn region and prevents binding of the Nrf2 peptide to Keap1.
Glutamate residues E79 and E82 are the only peptide residues whose side chains make specific interactions with the Kelch domain. The carboxylate oxygen atoms of E79 contact the side chains of Arg415, Arg483, and Ser508, while the carboxylate oxygen atoms of E82 make hydrogen bonds with the side chains of Ser363, Asn382, and Arg380. The peptide backbone makes five contacts with the Kelch domain, four from the carbonyl oxygen atoms of E78, E79, T80, and F83, and one from a backbone amide group of F83.
Solvent molecules mediate additional contacts between the peptide and the Kelch domain, enabling an additional contact between E79 and Arg415 and allowing E82 to interact with both Asn414 and Ser602. The carboxylate oxygen atom of D77 contacts two water molecules that make bridging hydrogen bonds with Arg415 and Arg380. The hydroxyl group of T80 utilizes a water molecule to interact with Arg380.
All six blades of the Kelch b-propeller contribute to complex formation. Kelch domain residues that contact the side chains of the peptide are concentrated on one side of the binding pocket in blades II, III, IV, and V, while residues that contact backbone atoms of the peptide or participate in van der Waals interactions are located on the other side of the binding pocket in blades V, VI, I and II.
Side chains from six residues in Keap1 (Ser363, Asn382, Arg380, Arg415, Arg483, and Ser508) participate in hydrogen bond interactions with the carboxylate oxygen atoms from E79 and E82 in the peptide. Side chains from five additional residues in Keap1 participate in hydrogen bonding with the peptide backbone: these are Tyr334, Asn382, Gln530, Ser555 and Ser602. Finally, the side chains from seven residues in Keap1 participate in van der Waals interactions with the peptide, including Tyr 334, Asn387, Arg415, Ser508, Tyr525, Tyr572 and Phe577. Functional analysis of mutant Keap1 proteins containing individual alanine substitutions indicates that charged residues and hydrophobic residues located in the binding pocket of Keap1 are the major contributors to the stability of the complex. In particular, the side chains of Tyr334, Arg380, Asn382, Arg415, Arg483, Tyr525, and Tyr572 in Keap1 each contact the Nrf2-derived peptide and individual alanine substitutions for these residues significantly impairs binding to Nrf2 and repression of Nrf2-dependent gene expression.
The human genome encodes more than 40 BTB-Kelch proteins. Point mutations in the Kelch domain of several BTB-Kelch proteins, including Keap1, gigaxonin and ENC1, have been associated with human diseases. In the case of Keap1, one gene variant (G364C) and one somatic mutation (G430C) have been associated with lung cancer. The G364C mutation is located in the A-D loop of blade I and likely disrupts the substrate binding pocket, while the G430C mutation is adjacent to the B strand in blade III and likely disrupts the structure of the Kelch domain. It is likely that disease-associated point mutations within the Kelch domains of both gigaxonin and ENC1 will perturb the association of gigaxonin and ENC1 with their respective substrate proteins. Furthermore, as the overall dimensions of the substrate binding pocket, formed by the D-A and the B-C loops, are likely to be very similar in other BTB-Kelch proteins, an intriguing suggestion is that the substrate(s) for these and other BTB-Kelch proteins may also utilize b-turn motifs to bind their cognate substrate adaptor.
Mark Hannink
 

Literature references that cite this PDB file's key reference

  PubMed id Reference
22266938 L.M.Boyden, M.Choi, K.A.Choate, C.J.Nelson-Williams, A.Farhi, H.R.Toka, I.R.Tikhonova, R.Bjornson, S.M.Mane, G.Colussi, M.Lebel, R.D.Gordon, B.A.Semmekrot, A.Poujol, M.J.Välimäki, M.E.De Ferrari, S.A.Sanjad, M.Gutkin, F.E.Karet, J.R.Tucci, J.R.Stockigt, K.M.Keppler-Noreuil, C.C.Porter, S.K.Anand, M.L.Whiteford, I.D.Davis, S.B.Dewar, A.Bettinelli, J.J.Fadrowski, C.W.Belsha, T.E.Hunley, R.D.Nelson, H.Trachtman, T.R.Cole, M.Pinsk, D.Bockenhauer, M.Shenoy, P.Vaidyanathan, J.W.Foreman, M.Rasoulpour, F.Thameem, H.Z.Al-Shahrouri, J.Radhakrishnan, A.G.Gharavi, B.Goilav, and R.P.Lifton (2012).
Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.
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21351160 F.Correa, E.Ljunggren, C.Mallard, M.Nilsson, S.G.Weber, and M.Sandberg (2011).
The Nrf2-inducible antioxidant defense in astrocytes can be both up- and down-regulated by activated microglia:Involvement of p38 MAPK.
  Glia, 59, 785-799.  
21414291 J.Zhao, J.B.Redell, A.N.Moore, and P.K.Dash (2011).
A novel strategy to activate cytoprotective genes in the injured brain.
  Biochem Biophys Res Commun, 407, 501-506.  
21365312 L.Baird, and A.T.Dinkova-Kostova (2011).
The cytoprotective role of the Keap1-Nrf2 pathway.
  Arch Toxicol, 85, 241-272.  
21370976 Z.Hua, and R.D.Vierstra (2011).
The cullin-RING ubiquitin-protein ligases.
  Annu Rev Plant Biol, 62, 299-334.  
20453876 E.Bobrovnikova-Marjon, C.Grigoriadou, D.Pytel, F.Zhang, J.Ye, C.Koumenis, D.Cavener, and J.A.Diehl (2010).
PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.
  Oncogene, 29, 3881-3895.  
20215646 G.P.Sykiotis, and D.Bohmann (2010).
Stress-activated cap'n'collar transcription factors in aging and human disease.
  Sci Signal, 3, re3.  
20446772 J.D.Hayes, M.McMahon, S.Chowdhry, and A.T.Dinkova-Kostova (2010).
Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway.
  Antioxid Redox Signal, 13, 1713-1748.  
20446770 J.Shlomai (2010).
Redox control of protein-DNA interactions: from molecular mechanisms to significance in signal transduction, gene expression, and DNA replication.
  Antioxid Redox Signal, 13, 1429-1476.  
19560482 K.R.Sekhar, G.Rachakonda, and M.L.Freeman (2010).
Cysteine-based regulation of the CUL3 adaptor protein Keap1.
  Toxicol Appl Pharmacol, 244, 21-26.  
20676377 N.G.Innamorato, A.Jazwa, A.I.Rojo, C.García, J.Fernández-Ruiz, A.Grochot-Przeczek, A.Stachurska, A.Jozkowicz, J.Dulak, and A.Cuadrado (2010).
Different susceptibility to the Parkinson's toxin MPTP in mice lacking the redox master regulator Nrf2 or its target gene heme oxygenase-1.
  PLoS One, 5, e11838.  
19951033 S.Rosales-Corral, R.J.Reiter, D.X.Tan, G.G.Ortiz, and G.Lopez-Armas (2010).
Functional aspects of redox control during neuroinflammation.
  Antioxid Redox Signal, 13, 193-247.  
20133743 T.Ogura, K.I.Tong, K.Mio, Y.Maruyama, H.Kurokawa, C.Sato, and M.Yamamoto (2010).
Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening, double glycine repeat, and C-terminal domains.
  Proc Natl Acad Sci U S A, 107, 2842-2847.  
20389280 Y.R.Lee, W.C.Yuan, H.C.Ho, C.H.Chen, H.M.Shih, and R.H.Chen (2010).
The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses.
  EMBO J, 29, 1748-1761.  
19726686 C.H.Gray, L.C.McGarry, H.J.Spence, A.Riboldi-Tunnicliffe, and B.W.Ozanne (2009).
Novel beta-propeller of the BTB-Kelch protein Krp1 provides a binding site for Lasp-1 that is necessary for pseudopodial extension.
  J Biol Chem, 284, 30498-30507.
PDB code: 2woz
19123792 H.Masutani, R.Otsuki, Y.Yamaguchi, M.Takenaka, N.Kanoh, K.Takatera, Y.Kunimoto, and J.Yodoi (2009).
Fragrant unsaturated aldehydes elicit activation of the Keap1/Nrf2 system leading to the upregulation of thioredoxin expression and protection against oxidative stress.
  Antioxid Redox Signal, 11, 949-962.  
19321346 J.D.Hayes, and M.McMahon (2009).
NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer.
  Trends Biochem Sci, 34, 176-188.  
19818708 M.Zhuang, M.F.Calabrese, J.Liu, M.B.Waddell, A.Nourse, M.Hammel, D.J.Miller, H.Walden, D.M.Duda, S.N.Seyedin, T.Hoggard, J.W.Harper, K.P.White, and B.A.Schulman (2009).
Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases.
  Mol Cell, 36, 39-50.
PDB codes: 3hqh 3hqi 3hql 3hqm 3hsv 3htm 3hu6 3hve 3ivq 3ivv
18618599 W.Li, and A.N.Kong (2009).
Molecular mechanisms of Nrf2-mediated antioxidant response.
  Mol Carcinog, 48, 91.  
18005231 A.I.Rojo, M.R.Sagarra, and A.Cuadrado (2008).
GSK-3beta down-regulates the transcription factor Nrf2 after oxidant damage: relevance to exposure of neuronal cells to oxidative stress.
  J Neurochem, 105, 192-202.  
18838122 A.Lau, N.F.Villeneuve, Z.Sun, P.K.Wong, and D.D.Zhang (2008).
Dual roles of Nrf2 in cancer.
  Pharmacol Res, 58, 262-270.  
  18391415 B.Padmanabhan, Y.Nakamura, and S.Yokoyama (2008).
Structural analysis of the complex of Keap1 with a prothymosin alpha peptide.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 64, 233-238.
PDB code: 2z32
18845810 C.A.Piantadosi, M.S.Carraway, A.Babiker, and H.B.Suliman (2008).
Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1.
  Circ Res, 103, 1232-1240.  
18057000 L.Li, M.Kobayashi, H.Kaneko, Y.Nakajima-Takagi, Y.Nakayama, and M.Yamamoto (2008).
Molecular Evolution of Keap1: TWO Keap1 MOLECULES WITH DISTINCTIVE INTERVENING REGION STRUCTURES ARE CONSERVED AMONG FISH.
  J Biol Chem, 283, 3248-3255.  
18353146 L.Marrot, C.Jones, P.Perez, and J.R.Meunier (2008).
The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis.
  Pigment Cell Melanoma Res, 21, 79-88.  
18200608 O.Okhrimenko, and I.Jelesarov (2008).
A survey of the year 2006 literature on applications of isothermal titration calorimetry.
  J Mol Recognit, 21, 1.  
18387606 S.C.Lo, and M.Hannink (2008).
PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to mitochondria.
  Exp Cell Res, 314, 1789-1803.  
18757741 T.Shibata, T.Ohta, K.I.Tong, A.Kokubu, R.Odogawa, K.Tsuta, H.Asamura, M.Yamamoto, and S.Hirohashi (2008).
Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy.
  Proc Natl Acad Sci U S A, 105, 13568-13573.  
17848967 B.D'Autréaux, and M.B.Toledano (2007).
ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.
  Nat Rev Mol Cell Biol, 8, 813-824.  
17785452 K.I.Tong, B.Padmanabhan, A.Kobayashi, C.Shang, Y.Hirotsu, S.Yokoyama, and M.Yamamoto (2007).
Different electrostatic potentials define ETGE and DLG motifs as hinge and latch in oxidative stress response.
  Mol Cell Biol, 27, 7511-7521.
PDB code: 2dyh
17049906 S.Prag, A.De Arcangelis, E.Georges-Labouesse, and J.C.Adams (2007).
Regulation of post-translational modifications of muskelin by protein kinase C.
  Int J Biochem Cell Biol, 39, 366-378.  
17046835 S.C.Lo, and M.Hannink (2006).
PGAM5, a Bcl-XL-interacting protein, is a novel substrate for the redox-regulated Keap1-dependent ubiquitin ligase complex.
  J Biol Chem, 281, 37893-37903.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.