4DLV

H-Ras Set 2 CaCl2/DTT, ordered off

Summary for 4DLV

• Entry DOI: 10.2210/pdb4dlv/pdb
• Related: 4DLR 4DLS 4DLT 4DLU 4DLW 4DLX 4DLY 4DLZ
• Descriptor: GTPase HRas, CALCIUM ION, MAGNESIUM ION, ... (6 entities in total)
• Functional Keywords: gtp-binding, nucleotide binding, signaling protein
• Biological source: Homo sapiens (human)
• Cellular location: Cell membrane. Isoform 2: Nucleus: P01112
• Total number of polymer chains: 1
• Total formula weight: 19665.62

Authors

Holzapfel, G.,Mattos, C. (deposition date: 2012-02-06, release date: 2012-08-08, Last modification date: 2024-02-28)

Primary citation

Holzapfel, G.,Buhrman, G.,Mattos, C.
Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition.
Biochemistry, 51:6114-6126, 2012

PubMed Abstract: Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

PubMed: 22845804
DOI: 10.1021/bi300509j

Experimental method

X-RAY DIFFRACTION (1.572 Å)