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PDBsum entry 1iaq

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protein ligands metals Protein-protein interface(s) links
Signaling protein PDB id
1iaq
Jmol
Contents
Protein chains
149 a.a. *
151 a.a. *
153 a.a. *
Ligands
GNP ×3
Metals
_MG ×3
* Residue conservation analysis
PDB id:
1iaq
Name: Signaling protein
Title: C-h-ras p21 protein mutant with thr 35 replaced by ser (t35s) complexed with guanosine-5'-[b,g-imido] triphosphate
Structure: Transforming protein p21/h-ras-1. Chain: a, b, c. Fragment: catalytic domain (residues 1-166). Synonym: c-h-ras. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: hras or hras1. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.90Å     R-factor:   0.242     R-free:   0.282
Authors: M.Spoerner,C.Herrmann,I.R.Vetter,H.R.Kalbitzer, A.Wittinghofer
Key ref:
M.Spoerner et al. (2001). Dynamic properties of the Ras switch I region and its importance for binding to effectors. Proc Natl Acad Sci U S A, 98, 4944-4949. PubMed id: 11320243 DOI: 10.1073/pnas.081441398
Date:
23-Mar-01     Release date:   06-Jun-01    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P01112  (RASH_HUMAN) -  GTPase HRas
Seq:
Struc:
189 a.a.
149 a.a.
Protein chain
Pfam   ArchSchema ?
P01112  (RASH_HUMAN) -  GTPase HRas
Seq:
Struc:
189 a.a.
151 a.a.
Protein chain
Pfam   ArchSchema ?
P01112  (RASH_HUMAN) -  GTPase HRas
Seq:
Struc:
189 a.a.
153 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   1 term 
  Biological process     signal transduction   3 terms 
  Biochemical function     GTP binding     1 term  

 

 
DOI no: 10.1073/pnas.081441398 Proc Natl Acad Sci U S A 98:4944-4949 (2001)
PubMed id: 11320243  
 
 
Dynamic properties of the Ras switch I region and its importance for binding to effectors.
M.Spoerner, C.Herrmann, I.R.Vetter, H.R.Kalbitzer, A.Wittinghofer.
 
  ABSTRACT  
 
We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)(.)GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. (31)P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. (31)P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.
 
  Selected figure(s)  
 
Figure 2.
Fig. 2. Structure of the nucleotide and effector region of Ras taken from the structure of Raps·GppNHp in complex with Raf-RBD (37). Ras is shown with van der Waals surface, highlighting the switch I region (red worm), with the indicated side chains and the guanine nucleotide in ball-and-stick representation. Mg2+ is shown as a yellow sphere. The image was prepared by using GRASP (49).
Figure 5.
Fig. 5. Kinetics of binding of Raf-RBD to Ras and Ras-mutants. 0.5 µM Ras complexed with mGppNHp was mixed with increasing concentrations of Raf-RBD in a stopped-flow apparatus, and the resulting pseudo-first-order reactions were measured (not shown). The observed pseudo-first-order rate constants are plotted against the concentrations of effector, as indicated, and the points are fitted to a two-step binding equation shown in the text. The resulting parameters are shown in Table 4.
 
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20949621 L.Gremer, T.Merbitz-Zahradnik, R.Dvorsky, I.C.Cirstea, C.P.Kratz, M.Zenker, A.Wittinghofer, and M.R.Ahmadian (2011).
Germline KRAS mutations cause aberrant biochemical and physical properties leading to developmental disorders.
  Hum Mutat, 32, 33-43.  
20457940 A.Zurita, Y.Zhang, L.Pedersen, T.Darden, and L.Birnbaumer (2010).
Obligatory role in GTP hydrolysis for the amide carbonyl oxygen of the Mg(2+)-coordinating Thr of regulatory GTPases.
  Proc Natl Acad Sci U S A, 107, 9596-9601.  
20124028 C.Pataki, T.Matusek, E.Kurucz, I.Andó, A.Jenny, and J.Mihály (2010).
Drosophila Rab23 is involved in the regulation of the number and planar polarization of the adult cuticular hairs.
  Genetics, 184, 1051-1065.  
20194776 G.Buhrman, G.Holzapfel, S.Fetics, and C.Mattos (2010).
Allosteric modulation of Ras positions Q61 for a direct role in catalysis.
  Proc Natl Acad Sci U S A, 107, 4931-4936.
PDB codes: 3k8y 3k9n 3lbh 3lbi 3lbn
20680402 K.Baskaran, K.Brunner, C.E.Munte, and H.R.Kalbitzer (2010).
Mapping of protein structural ensembles by chemical shifts.
  J Biomol NMR, 48, 71-83.  
20685651 S.Karassek, C.Berghaus, M.Schwarten, C.G.Goemans, N.Ohse, G.Kock, K.Jockers, S.Neumann, S.Gottfried, C.Herrmann, R.Heumann, and R.Stoll (2010).
Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.
  J Biol Chem, 285, 33979-33991.
PDB code: 2l0x
20170508 T.Zemojtel, M.Duchniewicz, Z.Zhang, T.Paluch, H.Luz, T.Penzkofer, J.S.Scheele, and F.J.Zwartkruis (2010).
Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity.
  BMC Evol Biol, 10, 55.  
19629046 A.Guilfoyle, M.J.Maher, M.Rapp, R.Clarke, S.Harrop, and M.Jormakka (2009).
Structural basis of GDP release and gating in G protein coupled Fe2+ transport.
  EMBO J, 28, 2677-2685.
PDB codes: 3hyr 3hyt
20059952 B.D.Slaughter, A.Das, J.W.Schwartz, B.Rubinstein, and R.Li (2009).
Dual modes of cdc42 recycling fine-tune polarized morphogenesis.
  Dev Cell, 17, 823-835.  
19300489 B.J.Grant, A.A.Gorfe, and J.A.McCammon (2009).
Ras conformational switching: simulating nucleotide-dependent conformational transitions with accelerated molecular dynamics.
  PLoS Comput Biol, 5, e1000325.  
19776012 C.Kiel, D.Filchtinski, M.Spoerner, G.Schreiber, H.R.Kalbitzer, and C.Herrmann (2009).
Improved binding of raf to Ras.GDP is correlated with biological activity.
  J Biol Chem, 284, 31893-31902.  
19651783 M.M.Edreira, S.Li, D.Hochbaum, S.Wong, A.A.Gorfe, F.Ribeiro-Neto, V.L.Woods, and D.L.Altschuler (2009).
Phosphorylation-induced conformational changes in Rap1b: allosteric effects on switch domains and effector loop.
  J Biol Chem, 284, 27480-27486.  
19240123 N.Tuncbag, G.Kar, O.Keskin, A.Gursoy, and R.Nussinov (2009).
A survey of available tools and web servers for analysis of protein-protein interactions and interfaces.
  Brief Bioinform, 10, 217-232.  
19394299 T.D.Bunney, O.Opaleye, S.M.Roe, P.Vatter, R.W.Baxendale, C.Walliser, K.L.Everett, M.B.Josephs, C.Christow, F.Rodrigues-Lima, P.Gierschik, L.H.Pearl, and M.Katan (2009).
Structural insights into formation of an active signaling complex between Rac and phospholipase C gamma 2.
  Mol Cell, 34, 223-233.
PDB codes: 2w2t 2w2v 2w2w 2w2x
18547521 A.A.Gorfe, B.J.Grant, and J.A.McCammon (2008).
Mapping the nucleotide and isoform-dependent structural and dynamical features of Ras proteins.
  Structure, 16, 885-896.  
18728185 D.W.Leung, C.Otomo, J.Chory, and M.K.Rosen (2008).
Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway.
  Proc Natl Acad Sci U S A, 105, 12797-12802.  
18713003 L.Gremer, B.Gilsbach, M.R.Ahmadian, and A.Wittinghofer (2008).
Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction.
  Biol Chem, 389, 1163-1171.  
18940599 M.B.Hamaneh, and M.Buck (2008).
Tripping a switch: PDZRhoGEF rgRGS-bound Galpha13.
  Structure, 16, 1439-1441.  
18348980 M.J.Phillips, G.Calero, B.Chan, S.Ramachandran, and R.A.Cerione (2008).
Effector proteins exert an important influence on the signaling-active state of the small GTPase Cdc42.
  J Biol Chem, 283, 14153-14164.
PDB code: 2qrz
18393397 M.Soundararajan, A.Turnbull, O.Fedorov, C.Johansson, and D.A.Doyle (2008).
RhoB can adopt a Mg2+ free conformation prior to GEF binding.
  Proteins, 72, 498-505.  
17189426 B.S.Shin, M.G.Acker, D.Maag, J.R.Kim, J.R.Lorsch, and T.E.Dever (2007).
Intragenic suppressor mutations restore GTPase and translation functions of a eukaryotic initiation factor 5B switch II mutant.
  Mol Cell Biol, 27, 1677-1685.  
17385754 C.Kötting, A.Kallenbach, Y.Suveyzdis, C.Eichholz, and K.Gerwert (2007).
Surface change of Ras enabling effector binding monitored in real time at atomic resolution.
  Chembiochem, 8, 781-787.  
17443350 J.L.Fuentes, K.Datta, S.M.Sullivan, A.Walker, and J.R.Maddock (2007).
In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.
  Mol Genet Genomics, 278, 105-123.  
17976838 J.L.Rudolph, G.X.Shi, E.Erdogan, A.P.Fields, and D.A.Andres (2007).
Rit mutants confirm role of MEK/ERK signaling in neuronal differentiation and reveal novel Par6 interaction.
  Biochim Biophys Acta, 1773, 1793-1800.  
17302736 M.Spoerner, A.Nuehs, C.Herrmann, G.Steiner, and H.R.Kalbitzer (2007).
Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPgammaS.
  FEBS J, 274, 1419-1433.  
16968776 C.Kötting, M.Blessenohl, Y.Suveyzdis, R.S.Goody, A.Wittinghofer, and K.Gerwert (2006).
A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein.
  Proc Natl Acad Sci U S A, 103, 13911-13916.  
17084704 S.Boykevisch, C.Zhao, H.Sondermann, P.Philippidou, S.Halegoua, J.Kuriyan, and D.Bar-Sagi (2006).
Regulation of ras signaling dynamics by Sos-mediated positive feedback.
  Curr Biol, 16, 2173-2179.  
16877502 S.O.Yesylevskyy, V.N.Kharkyanen, and A.P.Demchenko (2006).
The change of protein intradomain mobility on ligand binding: is it a commonly observed phenomenon?
  Biophys J, 91, 3002-3013.  
16147885 H.Zhi, W.Wang, L.Li, B.Chai, Y.Sun, and A.Liang (2005).
Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus.
  DNA Seq, 16, 260-265.  
16235217 M.Spoerner, T.F.Prisner, M.Bennati, M.M.Hertel, N.Weiden, T.Schweins, and H.R.Kalbitzer (2005).
Conformational states of human H-Ras detected by high-field EPR, ENDOR, and 31P NMR spectroscopy.
  Magn Reson Chem, 43, S74-S83.  
15197281 C.Kiel, T.Selzer, Y.Shaul, G.Schreiber, and C.Herrmann (2004).
Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex.
  Proc Natl Acad Sci U S A, 101, 9223-9228.  
14977045 E.W.Becker (2004).
Relevance of the kinetic equilibrium of forces to the control of the cell cycle by Ras proteins.
  Biol Chem, 385, 41-47.  
14648622 H.Gohlke, and D.A.Case (2004).
Converging free energy estimates: MM-PB(GB)SA studies on the protein-protein complex Ras-Raf.
  J Comput Chem, 25, 238-250.  
12581669 C.Herrmann (2003).
Ras-effector interactions: after one decade.
  Curr Opin Struct Biol, 13, 122-129.  
12878003 L.C.James, and D.S.Tawfik (2003).
Conformational diversity and protein evolution--a 60-year-old hypothesis revisited.
  Trends Biochem Sci, 28, 361-368.  
14556626 M.Geyer, C.Wilde, J.Selzer, K.Aktories, and H.R.Kalbitzer (2003).
Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy.
  Biochemistry, 42, 11951-11959.  
12869697 M.Sagermann, L.Gay, and B.W.Matthews (2003).
Long-distance conformational changes in a protein engineered by modulated sequence duplication.
  Proc Natl Acad Sci U S A, 100, 9191-9195.
PDB code: 1oyu
12717016 S.Kuppens, M.Hellings, J.Jordens, S.Verheyden, and Y.Engelborghs (2003).
Conformational states of the switch I region of Ha-ras-p21 in hinge residue mutants studied by fluorescence lifetime and fluorescence anisotropy measurements.
  Protein Sci, 12, 930-938.  
12437928 A.Doye, A.Mettouchi, G.Bossis, R.Clément, C.Buisson-Touati, G.Flatau, L.Gagnoux, M.Piechaczyk, P.Boquet, and E.Lemichez (2002).
CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion.
  Cell, 111, 553-564.  
12507428 B.S.Shin, D.Maag, A.Roll-Mecak, M.S.Arefin, S.K.Burley, J.R.Lorsch, and T.E.Dever (2002).
Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity.
  Cell, 111, 1015-1025.  
12093730 G.Buchwald, A.Friebel, J.E.Galán, W.D.Hardt, A.Wittinghofer, and K.Scheffzek (2002).
Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE.
  EMBO J, 21, 3286-3295.
PDB code: 1gzs
11839488 G.Schreiber (2002).
Kinetic studies of protein-protein interactions.
  Curr Opin Struct Biol, 12, 41-47.  
12069788 L.Columbus, and W.L.Hubbell (2002).
A new spin on protein dynamics.
  Trends Biochem Sci, 27, 288-295.  
11709168 K.Scheffzek, P.Grünewald, S.Wohlgemuth, W.Kabsch, H.Tu, M.Wigler, A.Wittinghofer, and C.Herrmann (2001).
The Ras-Byr2RBD complex: structural basis for Ras effector recognition in yeast.
  Structure, 9, 1043-1050.
PDB code: 1k8r
11709167 W.Gronwald, F.Huber, P.Grünewald, M.Spörner, S.Wohlgemuth, C.Herrmann, and H.R.Kalbitzer (2001).
Solution structure of the Ras binding domain of the protein kinase Byr2 from Schizosaccharomyces pombe.
  Structure, 9, 1029-1041.
PDB code: 1i35
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 codes are shown on the right.