Open AccessG-protein alpha o subunit: mutation of conserved cysteines identifies a subunitcontact surface and alters GDP affinity.
Author(s) -
Thomas C Thomas,
Carl J. Schmidt,
Eva J. Neer
Publication year - 1993
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.90.21.10295
Subject(s) - g alpha subunit , guanosine , interleukin 10 receptor, alpha subunit , protein subunit , cysteine , gamma subunit , alpha (finance) , binding site , g protein , beta (programming language) , chemistry , stereochemistry , biochemistry , receptor , enzyme , medicine , construct validity , nursing , computer science , programming language , patient satisfaction , gene
The reversible association of alpha and beta gamma subunits of GTP-bindingproteins is important for signal transmission from a variety of cell-surface receptors to intracellulareffectors. Previous work showed that 1,6-bis(maleimido)hexane, which crosslinks cysteine residues, crosslinksalpha o and alpha i-1 to beta gamma. These crosslinks are likely to form through a conserved cysteinebecause 1,6-bis(maleimido)hexane can also crosslink alpha i-2, alpha 1, alpha s and Drosophila alpha1 to give products of the same apparent molecular weight as crosslinked alpha o beta gamma and alphai-1 beta gamma. These proteins have only two cysteines in common. Therefore, we mutated each of the twoconserved cysteines of alpha o to alanines. Mutation of Cys215 prevents crosslinking to beta gamma, butdoes not affect binding of guanosine 5'-[gamma-thio]triphosphate or the ability of the mutated alphasubunit to bind beta gamma. In models of the alpha subunit based on the crystal structure of p21ras,Cys215 is located on the face opposite to the GTP-binding site and near an area that changes conformationdepending on the nucleotide bound. This surface on the alpha subunit overlaps a putative effector bindingregion, raising important questions about the spatial organization of the proteins as they form ternarycomplexes. Mutation of Cys325 has no effect on crosslinking but, surprisingly, decreases by a factorof 10 the affinity of the mutated protein for GDP, relative to wild type, without changing the affinityfor guanosine 5'-[gamma-thio]triphosphate. This mutation falls within a region thought to contact receptorsand may represent a site through which receptors enhance the release of GDP.