G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism.

The selective coupling of G protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here, we reveal the existence of a selectivity barcode (i.e. patterns of amino acids) on each of the 16 human G proteins that is recognised by distinct regions on the ~800 human receptors. Although universally conserved positions in the barcode allow the receptors to bind G proteins in a similar orientation, different receptors recognise the unique positions of the G protein barcode through distinct residues, similar to multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs permits the identification of these selectivity-determining residues. These findings lay the foundation for understanding the molecular basis of coupling selectivity within individual receptors and G proteins.Membrane protein receptors trigger the appropriate cellular response to extracellular stimuli by selective interaction with cytosolic adaptor proteins. In humans, GPCRs form the largest family of receptors with over 800 members1-3. Although GPCRs bind a staggering number of natural ligands (~1,000), they primarily couple to only four major Gα families encoded by 16 human genes3,4. Members of each of the four families regulate key effectors (e.g. adenylate cyclase, phospholipase C, etc.) and the generation of secondary messengers (e.g.

Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. Structural rearrangements of residue contacts in the transmembrane domain serve as 'activation pathways' that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a caseby-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.branched-chain amino acid pathway | cofactor imbalance K etol-acid reductoisomerases (KARI; EC 1.1.1.86) are a family of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductases that catalyze an alkyl-migration followed by a ketol-acid reduction of (S)-2-acetolactate (S2AL) and 2-aceto-2-hydroxybutyrate to yield (R)-2,3-dihydroxy-isovalerate and (R)-2,3-dihydroxy-3-methylvalerate, respectively (1), which are essential intermediates in the biosynthesis of branched-chain amino acids (BCAAs) (2, 3). The demand for these essential amino acids, used in the preparation of animal feed, human dietary supplements, and pharmaceuticals, is currently estimated to exceed 1,500 tons per year (4). In addition, the BCAA pathway has been engineered to produce fine chemicals and biofuels, including 1-butanol and isobutanol (5, 6). Under the anaerobic conditions preferred for large-scale fermentations, biosynthesis of BCAAs and other products that use this pathway is limited by the pathway's cofactor imbalance and reduced cellular production of NADPH (7,8). One approach to overcoming the cofactor imbalance is to engineer KARI to use NADH generated in glycolysis, thereby enabling anaerobic production of BCAA pathway products (7,8).Efforts to switch the cofactor specificity of oxidoreductases from NADPH to NADH have been made with varying degrees of success (8-17). The three reports of cofactor-switched KARIs (7,8,15) from two different organisms show few commonalities in terms of residues targeted for engineering. A general recipe for switching KARI cofactor specificity would allow metabolic engineers to take advantage of the natural sequence diversity of the KARI family, with concomitant diversity in properties such as expression level, pH tolerance, or thermal stability. By combining a systematic analysis of all reviewed and manually annotated [SwissProt (18)] KARIs, information from our previous work on switching the cofactor specificity of the Escherichia coli KARI (7), and available KARI structures, we have identified a subset of residues in ...

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