The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

A multitude of heptahelical receptors use heterotrimeric G proteins to transduce signals to specific effector target molecules. The G protein transducin, Gt, couples photon-activated rhodopsin with the effector cyclic GMP phosophodiesterase (PDE) in the vertebrate phototransduction cascade. The interactions of the Gt alpha-subunit (alpha(t)) with the inhibitory PDE gamma-subunit (PDEgamma) are central to effector activation, and also enhance visual recovery in cooperation with the GTPase-activating protein regulator of G-protein signalling (RGS)-9 (refs 1-3). Here we describe the crystal structure at 2.0 A of rod transducin alpha x GDP x AlF4- in complex with the effector molecule PDEgamma and the GTPase-activating protein RGS9. In addition, we present the independently solved crystal structures of the RGS9 RGS domain both alone and in complex with alpha(t/i1) x GDP x AlF4-. These structures reveal insights into effector activation, synergistic GTPase acceleration, RGS9 specificity and RGS activity. Effector binding to a nucleotide-dependent site on alpha(t) sequesters PDEgamma residues implicated in PDE inhibition, and potentiates recruitment of RGS9 for hydrolytic transition state stabilization and concomitant signal termination.

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an ␣ subunit with intrinsic GTPase activity and a ␤␥ heterodimer, the trimeric complex dissociates upon receptormediated nucleotide exchange on the ␣ subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the G␣ subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, G␣ subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein G␣ o in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon G␣ o binding, the determinants for GAP activity, and the structurally unique features of G␣ o that likely distinguish it physiologically from other members of the larger G␣ i family, affording insight to receptor, GAP and effector specificity.any extracellular cues ranging from photons to neurotransmitters are detected with high specificity by G proteincoupled receptors that in turn elicit an intracellular response by promoting GTP exchange on the ␣ subunit of a heterotrimeric G protein. The heterotrimeric G protein, composed of an ␣ subunit exhibiting endogenous GTPase activity and a heterodimeric ␤␥ subunit, dissociates, enabling each component to activate downstream effectors until GTP is hydrolyzed on the ␣ subunit and the heterotrimeric complex reforms. The ␣ subunit's endogenous GTP hydrolysis rate is relatively slow, therefore the cell uses GTPase accelerating proteins (GAPs) to increase the rate to suit the time scale and magnitude needed for a specific physiological response.The regulators of G protein signaling (RGS) proteins are a class of heterotrimeric G protein GAP first identified in Saccharomyces cerevisiae (Sst2) and Caenorhabditis elegans (Egl10) (1, 2). Studies, both biochemical and structural, have shown an overall preference for RGS domains to bind G␣ subunits in their transition state (mimicked by the analog GDP⅐AlF 4 Ϫ ) and to accelerate GTPase activity by stabilizing the transition state of hydrolysis, thereby optimizing the endogenous GTPase activity of the G␣ subunit without directly contributing to the hydrolytic mechanism (3-5). RGS proteins serve to quench the G protein signal temporally and spatially, either independently, or coupled (in cis or in trans) to an effector (6, 7). Thirty-seven RGS proteins have been identified in the human genome, cataloged into eight subfamilies based o...