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PDBsum entry 2k85

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protein links
Protein binding PDB id
2k85
Jmol
Contents
Protein chain
65 a.a. *
* Residue conservation analysis
PDB id:
2k85
Name: Protein binding
Title: P190-a rhogap ff1 domain
Structure: Glucocorticoid receptor DNA-binding factor 1. Chain: a. Fragment: unp residues 267-331. Synonym: glucocorticoid receptor repression factor 1, grf- 1, rho gap p190a, p190-a. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: grlf1, grf1, kiaa1722. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: contains a his-tag, a thioredoxin fusion protein and a tev cleavage-site
NMR struc: 15 models
Authors: R.Bonet,L.Ruiz,P.Martin-Malpartida,M.Macias
Key ref:
R.Bonet et al. (2009). NMR structural studies on human p190-A RhoGAPFF1 revealed that domain phosphorylation by the PDGF-receptor alpha requires its previous unfolding. J Mol Biol, 389, 230-237. PubMed id: 19393245 DOI: 10.1016/j.jmb.2009.04.035
Date:
02-Sep-08     Release date:   19-May-09    
PROCHECK
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 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q9NRY4  (RHG35_HUMAN) -  Rho GTPase-activating protein 35
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1499 a.a.
65 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain

 

 
DOI no: 10.1016/j.jmb.2009.04.035 J Mol Biol 389:230-237 (2009)
PubMed id: 19393245  
 
 
NMR structural studies on human p190-A RhoGAPFF1 revealed that domain phosphorylation by the PDGF-receptor alpha requires its previous unfolding.
R.Bonet, L.Ruiz, E.Aragón, P.Martín-Malpartida, M.J.Macias.
 
  ABSTRACT  
 
p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure. We found that RhoGAPFF1 does not fold with the typical (alpha1-alpha2-3(10)-alpha 3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an alpha1-alpha2-alpha 3-alpha 4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain. The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor alpha kinase form part of the alpha 3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction. Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.
 
  Selected figure(s)  
 
Figure 2.
Fig. 2. Comparison of RhoGAPFF1 with other FF domains. (a) Superimposition of the typical 3[10] helix (in red) of the FBP11FF1 domain (in dark blue) and the extended α helix (in yellow) that replaces the 3[10] helix in the RhoGAPFF1 domain (in sky blue). The residues that form part of this extended α helix are marked in a yellow box in the alignment in (b). (b) Sequence alignment of FF domains from human p190-A RhoGAP, CA150, and FBP11. The alignment was generated with ClustalX^16 and edited manually. The S symbol in a red box indicates the FF domain whose structure is deposited in the Protein Data Bank.^17 Conserved and semiconserved residues that form the hydrophobic core of the protein are indicated in green. Orange boxes mark the positions where one of the highly conserved Phe residues is substituted by another residue.
Figure 3.
Fig. 3. Tyr phosphorylation on RhoGAPFF1 domain. (a) Left: solid surface representation of the RhoGAPFF1 domain. The region corresponding to the consensus site (QDYVYL) for the kinase recognition is shown in green, and residues are labeled. The position of the hydroxyl group of Tyr308 is indicated in red. Right: detailed view of Tyr308 showing its buried side chain. (b) Matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) spectra of the phosphorylation assays for the RhoGAPFF1 domain (bottom) and for the positive control peptide DNEYFYV (top). Assays were performed in 50 μl of 10 mM Hepes (pH 7.5), 50 mM NaCl, 5 mM MgCl[2], 5 mM MnCl[2], 1.25 mM DTT, 0.2 mM ATP, 100 ng of PDGF receptor α kinase (Cell Signaling Technology®) and either 3 μg of protein or the peptide at a final concentration of 3 μM. Samples were collected at 303 or 310 K at a range of times, concentrated, and analyzed by MALDI-TOF mass spectrometry. Samples for the analysis were extracted before the reaction started (a), after 2 h at 303 K (b), and after 5 h at 310 K (c). (b and c) For the RhoGAPFF1 spectra, the region of the protein peaks has been zoomed to better appreciate the appearance of the peak corresponding to the phosphorylated domain [marked with an arrow in (c)].
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 389, 230-237) copyright 2009.  
  Figures were selected by the author.