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A constitutively active G? subunit provides insights into the mechanism of G protein activation.

ABSTRACT: The activation of G? subunits of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) is a critical event underlying a variety of biological responses. Understanding how G proteins are activated will require structural and biochemical analyses of GPCRs complexed to their G protein partners, together with structure-function studies of G? mutants that shed light on the different steps in the activation pathway. Previously, we reported that the substitution of a glycine for a proline at position 56 within the linker region connecting the helical and GTP-binding domains of a G? chimera, designated ?T*, yields a more readily exchangeable state for guanine nucleotides. Here we show that GDP-GTP exchange on ?T*(G56P), in the presence of the light-activated GPCR, rhodopsin (R*), is less sensitive to the ?1?1 subunit complex than to wild-type ?T*. We determined the X-ray crystal structure for the ?T*(G56P) mutant and found that the G56P substitution leads to concerted changes that are transmitted to the conformationally sensitive switch regions, the ?4-?6 loop, and the ?6 strand. The ?4-?6 loop has been proposed to be a GPCR contact site that signals to the TCAT motif and weakens the binding of the guanine ring of GDP, whereas the switch regions are the contact sites for the ?1?1 complex. Collectively, these biochemical and structural data lead us to suggest that ?T*(G56P) may be adopting a conformation that is normally induced within G? subunits by the combined actions of a GPCR and a G?? subunit complex during the G protein activation event.

SUBMITTER: Singh G

PROVIDER: S-EPMC3620018 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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A constitutively active Gα subunit provides insights into the mechanism of G protein activation.

Singh Garima G Ramachandran Sekar S Cerione Richard A RA

Biochemistry 20120405 15

The activation of Gα subunits of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) is a critical event underlying a variety of biological responses. Understanding how G proteins are activated will require structural and biochemical analyses of GPCRs complexed to their G protein partners, together with structure-function studies of Gα mutants that shed light on the different steps in the activation pathway. Previously, we reported that the substitution of a glycine for a proline at ...[more]

PMID: 22448927

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Project description:The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg(2+) bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerization of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving alpha1, loop beta5alpha5 and N terminus of alpha5 elements, also seen in the complex of PhoB RD with Mg(2+), in which helix alpha4 highly increases its flexibility. PhoB RD in complex with Mg(2+) and BeF(3) (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix alpha4 and shows another interaction involving alpha4, beta5 and alpha5 segments. We have selected a series of constitutively active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoB(CA) RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix alpha4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein.

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A constitutively active G? subunit provides insights into the mechanism of G protein activation.

Publications

A constitutively active Gα subunit provides insights into the mechanism of G protein activation.

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