1IAQ

C-H-RAS P21 PROTEIN MUTANT WITH THR 35 REPLACED BY SER (T35S) COMPLEXED WITH GUANOSINE-5'-[B,G-IMIDO] TRIPHOSPHATE

Summary for 1IAQ

• Entry DOI: 10.2210/pdb1iaq/pdb
• Descriptor: TRANSFORMING PROTEIN P21/H-RAS-1, MAGNESIUM ION, PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER (3 entities in total)
• Functional Keywords: gtp-binding, proto-oncogene, signaling protein
• Biological source: Homo sapiens (human)
• Cellular location: Cell membrane. Isoform 2: Nucleus: P01112
• Total number of polymer chains: 3
• Total formula weight: 58223.00

Authors

Spoerner, M.,Herrmann, C.,Vetter, I.R.,Kalbitzer, H.R.,Wittinghofer, A. (deposition date: 2001-03-23, release date: 2001-06-06, Last modification date: 2023-10-25)

Primary citation

Spoerner, M.,Herrmann, C.,Vetter, I.R.,Kalbitzer, H.R.,Wittinghofer, A.
Dynamic properties of the Ras switch I region and its importance for binding to effectors.
Proc.Natl.Acad.Sci.USA, 98:4944-4949, 2001

PubMed 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.

PubMed: 11320243
DOI: 10.1073/pnas.081441398

Experimental method

X-RAY DIFFRACTION (2.9 Å)